APP specific BACE inhibitors (ASBIs) and uses thereof

ABSTRACT

In certain embodiments APP-specific BACE inhibitors (ASBIs) are provided as well as uses thereof. In certain embodiments methods of preventing or delaying the onset of a pre-Alzheimer&#39;s condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre-Alzheimer&#39;s condition and/or cognitive dysfunction, or preventing or delaying the progression of a pre-Alzheimer&#39;s condition or cognitive dysfunction to Alzheimer&#39;s disease are provided where the method involves administering to a subject in need thereof an APP specific BACE inhibitor (ASBI) in an amount sufficient to prevent or delay the onset of a pre-Alzheimer&#39;s cognitive dysfunction, and/or to ameliorate one or more symptoms of a pre-Alzheimer&#39;s cognitive dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer&#39;s cognitive dysfunction to Alzheimer&#39;s disease. In certain embodiments the ASBI is a flavonoid (e.g. galangin) or flavonoid prodrug (e.g., galangin prodrug).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase of PCT/US2013/032481, filed onMar. 15, 2013, which claims benefit of and priority to U.S. Ser. No.61/728,688, filed on Nov. 20, 2012, and to U.S. Ser. No. 61/612,848,filed on Mar. 19, 2012, all of which are incorporated herein byreference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This work was supported in part by Grant No R01 AG034427 from NationalInstitute on Aging of the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND

Amyloid beta peptide (Aβ) is a primary component of beta amyloid fibrilsand plaques, which are regarded as having a role in an increasing numberof pathologies. Examples of such pathologies include, but are notlimited to, Alzheimer's disease, Down's syndrome, Parkinson's disease,memory loss (including memory loss associated with Alzheimer's diseaseand Parkinson's disease), attention deficit symptoms (includingattention deficit symptoms associated with Alzheimer's disease,Parkinson's disease, and Down's syndrome), dementia (includingpre-senile dementia, senile dementia, dementia associated withAlzheimer's disease, Parkinson's disease, and Down's syndrome),progressive supranuclear palsy, cortical basal degeneration,neurodegeneration, olfactory impairment (including olfactory impairmentassociated with Alzheimer's disease, Parkinson's disease, and Down'ssyndrome), β-amyloid angiopathy (including cerebral amyloid angiopathy),hereditary cerebral hemorrhage, mild cognitive impairment (“MCI”),glaucoma, amyloidosis, type II diabetes, hemodialysis (β microglobulinsand complications arising therefrom), neurodegenerative diseases such asscrapie, bovine spongiform encephalitis, Creutzfeld Jakob disease,traumatic brain injury and the like.

Aβ peptides are short peptides that are produced by proteolysis of thetransmembrane protein called amyloid precursor protein (“APP”). Aβpeptides are made from the cleavage of APP by β-secretase activity at aposition near the N-terminus of Aβ, and by gamma secretase activity at aposition near the C-terminus of Aβ. (APP is also cleaved by α-secretaseactivity, resulting in the secreted, non-amyloidogenic fragment known assoluble APPα). Beta site APP Cleaving Enzyme (“BACE-1”) is regarded asthe primary aspartyl protease responsible for the production of Aβ byβ-secretase activity. The inhibition of BACE-1 has been shown to inhibitthe production of Aβ.

Alzheimer's disease (AD) is estimated to afflict more than 20 millionpeople worldwide and is believed to be the most common cause ofdementia. As the World population ages, the number of people withAlzheimer's disease (AD, currently approximately 5.4 million in theUnited States, will continue to rise. Alzheimer's is a neurodegenerativedisease associated with progressive dementia and memory loss. Two keycharacteristics of AD are the accumulation of extracellular depositscontaining aggregated Aβ peptide and neuronal synaptic loss in the AD inspecific brain regions. Although AD pathogenesis is complex, compellinggenetic and biochemical evidence suggest that overproduction of Aβ, orfailure to clear this peptide is the earliest event in the amyloidcascade that lead to AD primarily through amyloid deposition, which ispresumed to be involved in neurofibrillary tangle formation, neuronaldysfunction and microglia activation, that characterize AD-affectedbrain tissues.

The accumulation of Aβ is considered to be the earliest event in acomplex cascade that leads to neurodegeneration, as discerned fromcompelling genetic and biochemical evidence. The amyloid cascadehypothesis (Hardy and Allsop (1991) Trends Pharmacol. Sci., 12: 383-388;Selkoe (1996) J. Biol. Chem., 271: 18295-18298; Hardy (1997) TrendsNeurosci., 20: 154-159; Hardy and Selkoe (2002) Science, 297: 353-356)states that overproduction of Aβ, or failure to clear this peptide,leads to AD, primarily through amyloid deposition, which is presumed tobe involved in neurofibrillary tangle formation, neuronal dysfunction,and microglia activation, that are hallmarks of AD-affected braintissues (Busciglio et al. (1995) Neuron, 14: 879-888; Gotz et al. (1995)EMBO J., 14: 1304-1313; Lewis et al. (2001) Science, 293: 1487-1491;Hardy et al. (1985) Nat Neurosci., 1: 355-358).

Considering the causative role of Aβ in AD etiology, novel therapeuticstrategies that lower Aβ levels or prevent the formation of theneurotoxic Aβ species have been suggested as a method to prevent or slowthe progression of the disease. Indeed, the major focus over the lastdecade has been to inhibit brain Aβ production and aggregation, toincrease parenchymal Aβ clearance, and to interfere with Aβ-induced celldeath.

The sequential cleavage of APP by membrane-bound proteases β-secretaseand γ-secretase results in the formation of Aβ. A competing proteolyticpathway to the β-secretase pathway, the α-secretase pathway, results incleavage of APP within the Aβ domain, thereby precluding the generationof Aβ (Selkoe (2001) Physiol. Rev., 81: 741-766; Hussain et al. (1999)Mol. Cell. Neurosci., 14: 419-427; Sinha et al. (1999) Nature, 402:537-540; Vassar et al. (1999) Science, 286: 735-741). The β-Site APPcleavage enzyme-1 (BACE1) was identified as the major β-secretaseactivity that mediates the first cleavage of APP in the β-amyloidogenicpathway (Id.).

BACE1 is a 501 amino acid protein that bears homology to eukaryoticaspartic proteases, especially from the pepsin family (Yan et al. (1999)Nature, 402: 533-537). Similar to other aspartic proteases, BACE1 issynthesized as a zymogen with a pro-domain that is cleaved by furin torelease the mature protein. BACE1 is a type-I transmembrane protein witha luminal active site that cleaves APP to release an ectodomain (sAPPβ)into the extracellular space. The remaining C-terminal fragment (CTF)undergoes further cleavage by γ-secretase, leading to the release of Aβand the APP intracellular C-terminal domain (AICD).

The presenilins have been proposed to be the major enzymatic componentof γ-secretase, whose imprecise cleavage of APP produces a spectrum ofAβ peptides varying in length by a few amino acids at the C-terminus.The majority of Aβ normally ends at amino acid 40 (Aβ40), but the42-amino acid variant (Aβ42) has been shown to be more susceptible toaggregation, and has been hypothesized to nucleate senile plaqueformation. The modulation of the γ-secretase can also lead to increasein the 38-amino acid variant (Aβ38). The competing α-secretase pathwayis the result of sequential cleavages by α- and γ-secretase. Threemetalloproteases of the disintegrin and metalloprotease family (ADAM 9,10, and 17) have been proposed as candidates for the α-secretaseactivity, which cleaves APP at position 16 within the Aβ sequence. Usingoverexpression experiments, ADAM-10 has been shown to be the likelyα-secretase for cleavage of APP (Vassar (2002) Adv. Drug Deliv. Rev.,54: 1589-1602; Buxbaum et al. (1998) J. Biol. Chem., 273: 27765-27767;Koike et al. (1999) Biochem. J., 343 (Pt 2): 371-375). This cleavagealso releases an ectodomain (sAPPα), which displays neuroprotectivefunctions (Lammich et al. (1999) Proc. Natl. Acad. Sci. USA, 96:3922-3927). Subsequent cleavage of the 83-amino acid CTF (C83) releasesp3, which is non-amyloidogenic, and AICD (Furukawa et al. (1996) J.Neurochem., 67: 1882-1896). The functions of these fragments are notfully elucidated, although AICD is hypothesized to mediate intracellularsignaling.

Research clarifying the metabolic pathways that regulate the productionof Aβ from the Amyloid Precursor Protein (APP) indicates that thesecretases that produce Aβ are good therapeutic targets, sinceinhibition of either β- or γ-secretase limits Aβ production. The factthat β-secretase initiates APP processing, and thus serves as the ratelimiting step in production of Aβ, its inhibition has attracted effortsby many research groups. Examples from the patent literature are growingand include, for example, WO2006009653, WO2007005404, WO2007005366,WO2007038271, WO2007016012, US2005/0282826, US2007072925, WO2007149033,WO2007145568, WO2007145569, WO2007145570, WO2007145571, WO2007114771,US20070299087, WO2005/016876, WO2005/014540, WO2005/058311,WO2006/065277, WO2006/014762, WO2006/014944, WO2006/138195,WO2006/138264, WO2006/138192, WO2006/138217, WO2007/050721,WO2007/053506, WO2007/146225, WO2006/138230, WO2006/138265,WO2006/138266, WO2007/053506, WO2007/146225, WO2008/073365,WO2008/073370, WO2008/103351, US2009/041201, US2009/041202, andWO2010/047372

A limitation of protease inhibitory strategies is the inhibition ofcleavage of all substrates of a given targeted protease, such as BACE orthe γ-secretase complex. In the case of γ-secretase, substrates otherthan APP, such as Notch, raise concerns for potential side effects ofγ-secretase inhibition, and the recent failure of the γ-secretaseinhibitor. Semagacestat, serves to reinforce such concerns.

BACE is a key enzyme involved in processing of APP leading to theproduction of Aβ42 and the Alzheimer's disease (AD) pathology. BACE-1(also called BACE) has become a popular research area since itsdiscovery, and has perhaps surpassed γ-secretase as the most promisingtarget for pharmaceutical research. One problem with γ-secretase as atarget is its known cleavage of Notch which serves important functionsin neuronal development. Presenilin knockout mice demonstrated abnormalsomitogenesis and axial skeletal development with shortened body length,as well as cerebral hemorrhages (Shen et al. (1997) Cell, 89: 629-639;Wong et al. (1997) Nature, 387: 288-292). In contrast, several groupsreported that BACE1 knockout mice are healthy and show no signs ofadverse effect (Luo et al. (2001) Nat. Neurosci., 4: 231-232; Roberds etal. (2001) Hum. Mol. Genet., 10: 1317-1324), while one group noticedsubtle neurochemical deficits and behavioral changes in otherwise viableand fertile mice (Harrison et al. (2003) Mol. Cell Neurosci., 24:646-655). Although recent studies have shown that BACE1 knockout miceexhibit hypomyelination of peripheral nerves (Willem et al. (2006)Science, 314: 664-666), the consequences of BACE1 inhibition in adultanimals, where myelination has already taken place, are unclear.Recently BACE1 has been reported to cleave multiple substrates,including ST6Gal I, PSGL-1, subunits of voltage-gated sodium channels,APP-like proteins (APLPs), LDL receptor related protein (LRP) and, mostrecently, type III neuregulin 1 (NRG1) (Willem et al. (2006) Science,314: 664-666; Hu et al. (2006) Nat. Neurosci., 9: 1520-1525). Theconsequences of inhibiting BACE1 directly are therefore not yet fullyunderstood.

Molecular modeling (Sauder et al. (2000) J. Mol. Biol., 300: 241-248)and subsequent X-ray crystallography (Hong et al. (2000) Science, 290:150-153; Maillard et al. (2007) J. Med. Chem., 50: 776-781) of theBACE-1 active site complexed with a transition-state inhibitor providedcrucial information about BACE-1-substrate interactions. Structurally,the BACE-1 active site is more open and less hydrophobic than otheraspartyl proteases, making development of effective in vivo BACEinhibitor candidates difficult. While a there is a large drug discoveryeffort focused on development of direct BACE inhibitors, none so farhave advanced significantly in clinical testing.

A few BACE inhibitors such as LY2811376 and CTS21166 entered clinicaltesting, but did not go forward beyond Phase-1 due to safety reasons.The discovery of other physiological substrates of BACE raises a majorconcern in the clinical development of BACE inhibitors or BACEmodulators and could be a significant roadblock in advancement of theseinhibitors as a therapy for the disease.

SUMMARY

In certain embodiments flavonoid and derivatives or analogues thereof(and prodrugs thereof) are identified that are believed to act asAPP-specific (or APP-selective) BACE inhibitors (ASBIs). In variousembodiments the flavonoids can be characterized by the formula:

where R¹ is selected from the group consisting of OH, O-saccaharide,O-alkyl, O-trifluoromethyl, O-aryl, O-heteroaryl, and carbamate; R⁴ andR⁵ are independently selected from the group consisting of H, OH, NH₂,O-alkyl, O-trifluoromethyl, S-alkyl, S-aryl, carboxylate, halogen,NH-alkyl, N,N-dialkyl, NHCO-alkyl, and heteroaryl, alkyl urea, andcarbamate; and R² and R³ are independently selected from the groupconsisting of H, OH, NH₂, O-alkyl, O-trifluoromethyl, S-alkyl, S-aryl,carboxylate, halogen, NH-alkyl, N,N-dialkyl, NHCO-alkyl, heteroaryl,alkyl urea, and carbamate. In certain embodiments R² and/or R³ is OH. Incertain embodiments R² is OH and R³ is OH. In certain embodiments R²and/or R³ are independently selected from the group consisting ofO-alkyl, S-alkyl, NH-alkyl and NHCO-alkyl. In certain embodiments R² andR³ are independently selected from the group consisting of O-alkyl,S-alkyl, NH-alkyl and NHCO-alkyl (e.g., where the alkyl component ofsaid O-alkyl, S-alkyl, NH-alkyl and NHCO-alkyl is a C₁₋₁₂ alkyl, or aC₁₋₉ alkyl, or a C₁₋₆ alkyl, or a C₁₋₃ alkyl). In certain embodiments R²and/or R³ is halogen (e.g., Cl, Br, Fl, I, etc.). In certain embodimentsR² is halogen and R³ is halogen. In certain embodiments R² and/or R³ areindependently selected from the group consisting of Cl, Br, Fl, and I.In certain embodiments R² and/or R³ is selected from the groupconsisting of S-aryl and heteroaryl. In certain embodiments R² and R³are independently selected S-aryl. In certain embodiments R² and R³ areindependently selected heteroaryl. In certain embodiments where R⁴and/or R⁵ is OH. In certain embodiments R⁴ is H and R⁵ is OH. In certainembodiments R⁴ is OH and R⁵ is H. In certain embodiments R⁴ is OH and R⁵is OH. In certain embodiments R⁴ and/or R⁵ is H. In certain embodimentsR⁴ is H and R⁵ is H. In certain embodiments when R⁴ and/or R⁵ is OH, R¹is O-Saccharide. In certain embodiments where R⁴ and/or R⁵ areindependently selected from the group consisting of O-alkyl, S-alkyl,NH-alkyl and NHCO-alkyl. In certain embodiments R⁴ and R⁵ areindependently selected from the group consisting of O-alkyl, S-alkyl,NH-alkyl and NHCO-alkyl (e.g., where the alkyl component of saidO-alkyl, S-alkyl, NH-alkyl and NHCO-alkyl is a C₁₋₁₂ alkyl, or a C₁₋₉alkyl, or a C₁₋₆ alkyl, or a C₁₋₃ alkyl). In certain embodiments R⁴and/or R⁵ is halogen. In certain embodiments R⁴ is halogen and R⁵ ishalogen. In certain embodiments R⁴ and/or R⁵ are independently selectedfrom the group consisting of Cl, Br, Fl, and I. In certain embodimentsR⁴ and/or R⁵ is selected from the group consisting of S-aryl andheteroaryl. In certain embodiments R⁴ and R⁵ are independently selectedS-aryl. In certain embodiments R⁴ and R⁵ are independently selectedheteroaryl. In certain embodiments R¹ is O-Saccharide (e.g.,O-monosaccharide, O-disaccharide, O-trisaccharide). In certainembodiments R¹ is O-alkyl, O-trifluoromethyl, O-aryl, or O-heteroaryl.In certain embodiments the APP specific (or selective) BACE inhibitor isgalangin or a derivative thereof. In certain embodiments the APPspecific BACE inhibitor is galangin. In certain embodiments the APPspecific BACE inhibitor is rutin or a derivative thereof. In certainembodiments the APP specific BACE inhibitor is rutin.

In various embodiments ASBI prodrugs are provided. In certainembodiments the prodrug is a galangin prodrug that is processed toglangin when administered to a mammal. Illustrative galangin prodrug(s)include, but are not limited to, a galangin prodrug characterized by theformula:

where R¹, R², and R³ are H, or a protecting group that is removed invivo in a mammal, where at least one of R¹, R², and R³ is not H; andwhere the prodrug partially or completely inhibits BACE processing ofAPP when administered to a mammal. In certain embodiments least one ofR¹, R², and R³ are independently selected from the group consisting of

In certain embodiments R¹ is H and R² and R³ are the same or differentand comprise any combination of the groups shown above. In certainembodiments the prodrug has a formula shown in FIG. 1 and/or FIG. 2.

In certain embodiments, the ASBI and/or ASBI prodrug is provided as apharmaceutical formulation where the ASBI and/or ASBI prodrug is theprinciple active component. In certain embodiments the ASBI and/or ASBIprodrug is the sole pharmaceutically active component (e.g., where thepharmaceutical activity is inhibition of BACE). In certain embodimentsthe ASBI and/or ASBI prodrug is provided in a pharmaceutical formulationwhere there is no other component is provided for neuropharmacologicalor neuropsychiatric activity.

In certain embodiments methods of preventing or delaying the onset of apre-Alzheimer's condition and/or cognitive dysfunction, and/orameliorating one or more symptoms of a pre-Alzheimer's condition and/orcognitive dysfunction, or preventing or delaying the progression of apre-Alzheimer's condition or cognitive dysfunction to Alzheimer'sdisease, are provided. The methods typically comprise administering (orcausing to be administered) to a subject in need thereof an APP specificBACE inhibitor (ASBI) and/or ASBI prodrug in an amount sufficient toprevent or delay the onset of a pre-Alzheimer's cognitive dysfunction,and/or to ameliorate one or more symptoms of a pre-Alzheimer's cognitivedysfunction, and/or to prevent or delay the progression of apre-Alzheimer's cognitive dysfunction to Alzheimer's disease. In certainembodiments the ASBI and/or ASBI prodrug comprises an ASBI flavonoidand/or an ASBI prodrug as described herein (e.g., as described above).In certain embodiments the ASBI is galangin and/or the ASBI prodrug is agalangin prodrug. In certain embodiments the ASBI and/or ASBI prodrug(e.g., galangin and/or galangin prodrug) is administered in apharmaceutical formulation wherein the ASBI is the principle activecomponent. In certain embodiments the ASBI and/or ASBI prodrug (e.g.,galangin and/or galangin prodrug) is administered in a pharmaceuticalformulation where the ASBI and/or the ASBI prodrug is the solepharmaceutically active component. In certain embodiments the ASBIand/or ASBI prodrug (e.g., galangin and/or galangin prodrug) isadministered in a pharmaceutical formulation and no other agent isprovided for neuropharmacological or neuropsychiatric activity. Incertain embodiments the method is a method of preventing or delaying thetransition from a cognitively asymptomatic pre-Alzheimer's condition toa pre-Alzheimer's cognitive dysfunction. In certain embodiments themethod is a method of preventing or delaying the onset of apre-Alzheimer's cognitive dysfunction. In certain embodiments the methodcomprises ameliorating one or more symptoms of a pre-Alzheimer'scognitive dysfunction. In certain embodiments the method comprisespreventing or delaying the progression of a pre-Alzheimer's cognitivedysfunction to Alzheimer's disease. In certain embodiments the subjectexhibits biomarker positivity of Aβ in a clinically normal human subjectage 50 or older, or 55 or older, or 60 or older, or 65 or older, or 70or older, or 75 or older, or 80 or older. In certain embodiments thesubject exhibits asymptomatic cerebral amyloidosis. In certainembodiments the subject exhibits cerebral amyloidosis in combinationwith downstream neurodegeneration. In certain embodiments the subjectexhibits cerebral amyloidosis in combination with downstreamneurodegeneration and subtle cognitive/behavioral decline. In certainembodiments the downstream neurodegeneration is determined by one ormore elevated markers of neuronal injury selected from the groupconsisting of tau, and FDG uptake. In certain embodiments the cerebralamyloidosis is determined by PET, or CSF analysis, and/or structural MRI(sMRI). In certain embodiments the subject is a subject diagnosed withmild cognitive impairment. In certain embodiments the subject shows aclinical dementia rating above zero and below about 1.5. In certainembodiments the subject is human. In certain embodiments the subject isat risk of developing Alzheimer's disease. In certain embodiments thesubject has a familial risk for having Alzheimer's disease. In certainembodiments the has a familial Alzheimer's disease (FAD) mutation. Incertain embodiments the subject has the APOE ε4 allele. In certainembodiments the administration of the ASBI and/or ASBI prodrug delays orprevents the progression of MCI to Alzheimer's disease. In certainembodiments the subject is free of and does not have genetic riskfactors of Parkinson's disease or schizophrenia. In certain embodimentsthe subject is not diagnosed as having or at risk for Parkinson'sdisease or schizophrenia. In certain embodiments the subject is notdiagnosed as at risk for a neurological disease or disorder other thanAlzheimer's disease. In certain embodiments the administration producesa reduction in the CSF of levels of one or more components selected fromthe group consisting of total-Tau (tTau), phospho-Tau (pTau), APPneo,soluble Aβ40, pTau/Aβ42 ratio and tTau/Aβ42 ratio, and/or an increase inthe CSF of levels of one or more components selected from the groupconsisting of Aβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβratio, sAPPα/Aβ40 ratio, and sAPPα/Aβ42 ratio. In certain embodimentsthe administration produces a reduction of the plaque load in the brainof the subject. In certain embodiments the administration produces areduction in the rate of plaque formation in the brain of the subject.In certain embodiments the administration produces an improvement in thecognitive abilities of the subject. In certain embodiments theadministration produces an improvement in, a stabilization of, or areduction in the rate of decline of the clinical dementia rating (CDR)of the subject. In certain embodiments the subject is a human and theadministration produces a perceived improvement in quality of life bythe human. In certain embodiments the ASBI and/or ASBI prodrug isadministered via a route selected from the group consisting of oraldelivery, isophoretic delivery, transdermal delivery, parenteraldelivery, aerosol administration, administration via inhalation,intravenous administration, topical administration to the eye,intraocular injection, and rectal administration. In certain embodimentsthe compound is administered orally. In certain embodiments theadministering is over a period of at least three weeks, or over a periodof at least 6 months, or over a period of at least one year. In certainembodiments the ASBI and/or ASBI prodrug is formulated foradministration via a route selected from the group consisting ofisophoretic delivery, transdermal delivery, aerosol administration,administration via inhalation, oral administration, intravenousadministration, topical delivery to the eye, intraocular injection, andrectal administration. In certain embodiments the anacetylcholinesterase inhibitor (e.g., tacrineipidacrine, galantamine,donepezil, icopezil, zanapezil, rivastigmine, Namenda, huperzine A,phenserine, physostigmine, neostigmine, pyridostigmine, ambenonium,demarcarium, edrophonium, ladostigil and ungeremine, metrifonate, etc.)is not administered in conjunction with the ASBI and/or ASBI prodrug.

In various embodiments methods of ameliorating one or more symptoms ofAlzheimer's disease, and/or reversing Alzheimer's disease, and/orreducing the rate of progression of Alzheimer's disease, are provided.The methods typically comprise administering to a subject in needthereof (or causing to be administered) an APP specific BACE inhibitor(ASBI) and/or an ASBI prodrug in an amount sufficient to ameliorate oneor more symptoms of Alzheimer's disease, and/or to reverse Alzheimer'sdisease, and/or to reduce the rate of progression of Alzheimer'sdisease. In certain embodiments the ASBI and/or ASBI prodrug comprisesan ASBI flavonoid and/or an ASBI prodrug as described herein (e.g., asdescribed above). In certain embodiments the ASBI is galangin and/or theASBI prodrug is a galangin prodrug (e.g., a prodrug shown in FIG. 1and/or FIG. 2). In certain embodiments the ASBI and/or ASIB prodrug isadministered in a pharmaceutical formulation where the ASBI and/or ASBIprodrug is the principle active component. In certain embodiments theASBI and/or ASBI prodrug is administered in a pharmaceutical formulationwhere the ASBI and/or ASBI prodrug is the sole pharmaceutically activecomponent. In certain embodiments the ASBI and/or ASBI prodrug isadministered in a pharmaceutical formulation no other component isprovided for neuropharmacological or neuropsychiatric activity. Incertain embodiments the subject is a human (or a non-human mammal). Incertain embodiments the subject is a human age 50 or older, or 55 orolder, or 60 or older, or 65 or older, or 70 or older, or 75 or older,or 80 or older. In certain embodiments the subject is diagnosed withearly stage Alzheimer's disease. In certain embodiments the subject isdiagnosed with mid-stage Alzheimer's disease. In certain embodiments thesubject is diagnosed with late-stage Alzheimer's disease. In certainembodiments the administering reduces the severity of Alzheimer'sdisease. In certain embodiments the administering ameliorates one ormore symptoms of Alzheimer's disease. In certain embodiments theadministering reduces the rate of progression of Alzheimer's disease. Incertain embodiments the administering results in a reduction in the CSFof levels of one or more components selected from the group consistingof total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Aβ40, pTau/Aβ42ratio and tTau/Aβ42 ratio, and/or an increase in the CSF of levels ofone or more components selected from the group consisting of Aβ42/Aβ40ratio, Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, andsAPPα/Aβ42 ratio. is a method of preventing or delaying the transitionfrom a cognitively asymptomatic pre-Alzheimer's condition to apre-Alzheimer's cognitive dysfunction. In certain embodiments theadministration produces a reduction of the plaque load in the brain ofthe subject. In certain embodiments the administration produces areduction in the rate of plaque formation in the brain of the subject.In certain embodiments the administration produces an improvement in thecognitive abilities of the subject. In certain embodiments theadministration produces an improvement in, a stabilization of, or areduction in the rate of decline of the clinical dementia rating (CDR)of the subject. In certain embodiments the subject is a human and saidadministration produces a perceived improvement in quality of life bythe human. In certain embodiments the administering results in reducedcerebral amyloidosis and/or downstream neurodegeneration. In certainembodiments the downstream neurodegeneration is determined by one ormore markers of neuronal injury selected from the group consisting oftau, FDG uptake, decrease in sAPPalpha, increase in sAPPbeta, and Abeta.In certain embodiments the cerebral amyloidosis is determined by PET,CSF analysis. and structural MRI (sMRI). In certain embodiments thesubject shows a clinical dementia rating indicative of Alzheimer'sdisease. In certain embodiments the subject has a familial risk forhaving Alzheimer's disease. In certain embodiments the subject has afamilial Alzheimer's disease (FAD) mutation. In certain embodiments thesubject has the APOE ε4 allele. In certain embodiments the subject isfree of and does not have genetic risk factors of Parkinson's disease orschizophrenia. In certain embodiments the subject is not diagnosed ashaving or at risk for Parkinson's disease or schizophrenia. In certainembodiments the subject does not have a neurological disease or disorderother than Alzheimer's disease. In certain embodiments the subject isnot diagnosed as having or at risk for a neurological disease ordisorder other than Alzheimer's disease. In certain embodiments the ASBIand/or ASBI prodrug is administered via a route selected from the groupconsisting of oral delivery, isophoretic delivery, transdermal delivery,parenteral delivery, aerosol administration, administration viainhalation, intravenous administration, subcutaneous administration,topical administration to the eye, intraocular injection, and rectaladministration. In certain embodiments the ASBI and/or ASBI prodrug isformulated for administration via a route selected from the groupconsisting of oral delivery, isophoretic delivery, transdermal delivery,parenteral delivery, aerosol administration, administration viainhalation, intravenous administration, and rectal administration. Incertain embodiments the ASBI and/or ASBI prodrug is administered orally.In certain embodiments the administering is over a period of at leastthree weeks, or over a period of at least 6 months, or over a period ofat least one year. In certain embodiments an acetylcholinesteraseinhibitor (e.g., tacrineipidacrine, galantamine, donepezil, icopezil,zanapezil, rivastigmine, Namenda, huperzine A, phenserine,physostigmine, neostigmine, pyridostigmine, ambenonium, demarcarium,edrophonium, ladostigil and ungeremine, metrifonate, and the like) isnot administered in conjunction with said ASBI and/or ASBI prodrug. Incertain embodiments the ASBI and/or ASBI prodrug is administered via aroute selected from the group consisting of oral delivery, isophoreticdelivery, transdermal delivery, parenteral delivery, aerosoladministration, administration via inhalation, intravenousadministration, and rectal administration.

In various embodiments methods of slowing the progression, stopping, orreversing age-related macular degeneration (AMD) and/or glaucoma in amammal are provided. The methods typically involve administering to themammal an ASBI and/or an ASBI prodrug in an amount sufficient to slowthe progression, to stop, or to reverse, and/or to ameliorate one ormore symptomse and/or markers of said AMD and/or glaucoma. In certainembodiments the ASBI and/or ASBI prodrug comprises an ASBI flavonoidand/or an ASBI prodrug as described herein (e.g., as described above).In certain embodiments the ASBI is galangin and/or the ASBI prodrug is agalangin prodrug (e.g., a prodrug shown in FIG. 1 and/or FIG. 2). Incertain embodiments the ASBI and/or ASBI prodrug is administered via aroute selected from the group consisting of oral delivery, isophoreticdelivery, transdermal delivery, parenteral delivery, aerosoladministration, administration via inhalation, intravenousadministration, subcutaneous administration, topical administration tothe eye, intraocular injection, and rectal administration. In certainembodiments the ASBI and/or ASBI prodrug is formulated foradministration via a route selected from the group consisting of oraldelivery, isophoretic delivery, transdermal delivery, parenteraldelivery, aerosol administration, administration via inhalation,intravenous administration, and rectal administration. In certainembodiments the ASBI and/or ASBI prodrug is administered to the eye(e.g., via eye drops, intraocular injection, and the like). In certainembodiments the administering is over a period of at least three weeks,or over a period of at least 6 months, or over a period of at least oneyear.

Definitions

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Accordingly, isotopically labeled compounds are within thescope of this invention.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup will be substituted with one or more substituents, unlessotherwise specified. In some embodiments, a substituted group issubstituted with 1, 2, 3, 4, 5, or 6 substituents. Examples ofsubstituent groups include: halogens (i.e., F, Cl, Br, and I);hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy,heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines;aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls;sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones;azides; amides; ureas; amidines; guanidines; enamines; imides;isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitrogroups; nitriles (i.e., CN), and the like.

The term “alkyl” refers to and covers any and all groups that are knownas normal alkyl, branched-chain alkyl, cycloalkyl and alsocycloalkyl-alkyl. Illustrative alkyl groups include, but are not limitedto methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,t-butyl, octyl, and decyl. The term “cycloalkyl” refers to cyclic,including polycyclic, saturated hydrocarbyl groups. Examples include,but are not limited to cyclopentyl, cyclohexyl, dicyclopentyl,norbornyl, octahydronapthyl, and spiro[3.4]octyl. In certainembodiments, alkyl groups contain 1-12 carbon atoms (C1-12 alkyl), or1-9 carbon atoms (C₁₋₉ alkyl), or 1-6 carbon atoms (C₁₋₆ alkyl), or 1-5carbon atoms (C₁₋₅ alkyl), or carbon atoms (C₁₋₄ alkyl), or 1-3 carbonatoms (C₁₋₃ alkyl), or 1-2 carbon atoms (C₁₋₂ alkyl).

By way of example, the term “C₁₋₆ alkyl group” refers to a straightchain or branched chain alkyl group having 1 to 6 carbon atoms, and maybe exemplified by a methyl group, an ethyl group, an n-propyl group, anisopropyl group, an n-butyl group, an isobutyl group, a tert-butylgroup, a sec-butyl group, an n-pentyl group, a tert-amyl group, a3-methylbutyl group, a neopentyl group, and an n-hexyl group.

The term “alkoxy” as used herein means an alkyl group bound through asingle, terminal oxygen atom. An “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. The term “aryloxy” is used ina similar fashion, and may be represented as —O-aryl, with aryl asdefined below. The term “hydroxy” refers to —OH.

Similarly, the term “alkylthio” as used herein means an alkyl groupbound through a single, terminal sulfur atom. An “alkylthio” group maybe represented as —S-alkyl where alkyl is as defined above. The term“arylthio” is used similarly, and may be represented as —S-aryl, witharyl as defined below. The term “mercapto” refers to —SH.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups include monocyclic, bicyclic and polycyclicring systems. Thus, aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl,indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments,aryl groups contain 6-14 carbons, and in others from 6 to 12 or even6-10 carbon atoms in the ring portions of the groups. Although thephrase “aryl groups” includes groups containing fused rings, such asfused aromatic-aliphatic ring systems (e.g., indanyl,tetrahydronaphthyl, and the like), it does not include aryl groups thathave other groups, such as alkyl or halo groups, bonded to one of thering members. Rather, groups such as tolyl are referred to assubstituted aryl groups. Representative substituted aryl groups may bemono-substituted or substituted more than once. For example,monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-,5-, or 6-substituted phenyl or naphthyl groups, which may be substitutedwith substituents such as those listed above.

The term “heteroaryl group” refers to a monocyclic or condensed-ringaromatic heterocyclic group containing one or more hetero-atoms selectedfrom O, S and N. If the aromatic heterocyclic group has a condensedring, it can include a partially hydrogenated monocyclic group. Examplesof such a heteroaryl group include a pyrazolyl group, a thiazolyl group,an isothiazolyl group, a thiadiazolyl group, an imidazolyl group, afuryl group, a thienyl group, an oxazolyl group, an isoxazolyl group, apyrrolyl group, an imidazolyl group, a (1,2,3)- and (1,2,4)-triazolylgroup, a tetrazolyl group, a pyranyl group, a pyridyl group, apyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a quinolylgroup, an isoquinolyl group, a benzofuranyl group, an isobenzofuranylgroup, an indolyl group, an isoindolyl group, an indazolyl group, abenzoimidazolyl group, a benzotriazolyl group, a benzoxazolyl group, abenzothiazolyl group, a benzo[b]thiophenyl group, athieno[2,3-b]thiophenyl group, a (1,2)- and (1,3)-benzoxathiol group, achromenyl group, a 2-oxochromenyl group, a benzothiadiazolyl group, aquinolizinyl group, a phthalazinyl group, a naphthyridinyl group, aquinoxalinyl group, a quinazolinyl group, a cinnolinyl group, and acarbazolyl group.

A “derivative” of a compound means a chemically modified compoundwherein the chemical modification takes place at one or more functionalgroups of the compound. The derivative however, is expected to retain,or enhance, the pharmacological activity of the compound from which itis derived.

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, pulmonary, andtopical/transdermal administration. Routes of administration for agents(e.g., ASBIs such as galangin, rutin, and analogues, derivatives, orprodrugs thereof, or tautomer(s) or stereoisomer(s) thereof, orpharmaceutically acceptable salts or solvates of said ASBI(s), saidstereoisomer(s), or said tautomer(s), or analogues, derivatives, orprodrugs thereof) that find use in the methods described herein include,e.g., oral (per os (p.o.)) administration, nasal or inhalationadministration, administration as a suppository, topical contact,transdermal delivery (e.g., via a transdermal patch), intrathecal (IT)administration, intravenous (“iv”) administration, intraperitoneal(“ip”) administration, intramuscular (“im”) administration,intralesional administration, or subcutaneous (“sc”) administration, orthe implantation of a slow-release device e.g., a mini-osmotic pump, adepot formulation, etc., to a subject. Administration can be by anyroute including parenteral and transmucosal (e.g., oral, nasal, vaginal,rectal, or transdermal). Parenteral administration includes, e.g.,intravenous, intramuscular, intra-arterial, intradermal, subcutaneous,intraperitoneal, intraventricular, ionophoretic and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “systemic administration” and “systemically administered”refer to a method of administering the agent(s) described herein orcomposition to a mammal so that the agent(s) or composition is deliveredto sites in the body, including the targeted site of pharmaceuticalaction, via the circulatory system. Systemic administration includes,but is not limited to, oral, intranasal, rectal and parenteral (e.g.,other than through the alimentary tract, such as intramuscular,intravenous, intra-arterial, transdermal and subcutaneous)administration.

The term “co-administering” or “concurrent administration” or“administering in conjunction with” when used, for example with respectto the active agent(s) described herein e.g., ASBIs such as galangin,rutin, and analogues, derivatives, or prodrugs thereof) and a secondactive agent (e.g., a cognition enhancer), refers to administration ofthe agent(s) and/the second active agent such that both cansimultaneously achieve a physiological effect. The two agents, however,need not be administered together. In certain embodiments,administration of one agent can precede administration of the other.Simultaneous physiological effect need not necessarily require presenceof both agents in the circulation at the same time. However, in certainembodiments, co-administering typically results in both agents beingsimultaneously present in the body (e.g., in the plasma) at asignificant fraction (e.g., 20% or greater, preferably 30% or 40% orgreater, more preferably 50% or 60% or greater, most preferably 70% or80% or 90% or greater) of their maximum serum concentration for anygiven dose.

The term “effective amount” or “pharmaceutically effective amount” referto the amount and/or dosage, and/or dosage regime of one or moreagent(s) necessary to bring about the desired result e.g., an amountsufficient to mitigating in a mammal one or more symptoms associatedwith mild cognitive impairment (MCI), or an amount sufficient to lessenthe severity or delay the progression of a disease characterized byamyloid deposits in the brain in a mammal (e.g., therapeuticallyeffective amounts), an amount sufficient to reduce the risk or delayingthe onset, and/or reduce the ultimate severity of a diseasecharacterized by amyloid deposits in the brain in a mammal (e.g.,prophylactically effective amounts).

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s) at issue to the subject. Causing to be administered caninvolve diagnosis and/or determination of an appropriate therapeutic orprophylactic regimen, and/or prescribing particular agent(s) for asubject. Such prescribing can include, for example, drafting aprescription form, annotating a medical record, and the like.

As used herein, the terms “treating” and “treatment” refer to delayingthe onset of, retarding or reversing the progress of, reducing theseverity of, or alleviating or preventing either the disease orcondition to which the term applies, or one or more symptoms of suchdisease or condition.

The term “mitigating” refers to reduction or elimination of one or moresymptoms of that pathology or disease, and/or a reduction in the rate ordelay of onset or severity of one or more symptoms of that pathology ordisease, and/or the prevention of that pathology or disease. In certainembodiments, the reduction or elimination of one or more symptoms ofpathology or disease can include, but is not limited to, reduction orelimination of one or more markers that are characteristic of thepathology or disease (e.g., of total-Tau (tTau), phospho-Tau (pTau),APPneo, soluble Aβ40, pTau/Aβ42 ratio and tTau/Aβ42 ratio, and/or anincrease in the CSF of levels of one or more components selected fromthe group consisting of Aβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα,sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, sAPPα/Aβ42 ratio, etc.) and/orreduction, stabilization or reversal of one or more diagnostic criteria(e.g., clinical dementia rating (CDR)).

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents recited in a method orcomposition, and further can include other agents that, on their own donot substantial activity for the recited indication or purpose. In someembodiments, the phrase “consisting essentially of” expressly excludesthe inclusion of one or more additional agents that haveneuropharmacological activity other than the recited agent(s) (e.g.,other than ASBIs such as galangin, rutin, and analogues, derivatives, orprodrugs thereof). In some embodiments, the phrase “consistingessentially of” expressly excludes the inclusion of one or moreadditional active agents other than the active agent(s) described herein(e.g., other than ASBIs such as galangin, rutin, and analogues,derivatives, or prodrugs thereof). In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore acetylcholinesterase inhibitors.

The terms “subject,” “individual,” and “patient” interchangeably referto a mammal, preferably a human or a non-human primate, but alsodomesticated mammals (e.g., canine or feline), laboratory mammals (e.g.,mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g.,equine, bovine, porcine, ovine). In various embodiments, the subject canbe a human (e.g., adult male, adult female, adolescent male, adolescentfemale, male child, female child) under the care of a physician or otherhealthworker in a hospital, psychiatric care facility, as an outpatient,or other clinical context. In certain embodiments the subject may not beunder the care or prescription of a physician or other healthworker.

The term “formulation” or “drug formulation” or “dosage form” or“pharmaceutical formulation” as used herein refers to a compositioncontaining at least one therapeutic agent or medication for delivery toa subject. In certain embodiments the dosage form comprises a given“formulation” or “drug formulation” and may be administered to a patientin the form of a lozenge, pill, tablet, capsule, suppository, membrane,strip, liquid, patch, film, gel, spray or other form.

The term “mucosal membrane” refers generally to any of the mucus-coatedbiological membranes in the body. In certain embodiments active agent(s)described herein can be administered herein via any mucous membranefound in the body, including, but not limited to buccal, perlingual,nasal, sublingual, pulmonary, rectal, and vaginal mucosa. Absorptionthrough the mucosal membranes of the oral cavity and those of the gutare of interest. Thus, peroral, buccal, sublingual, gingival and palatalabsorption are contemplated herein.

The term “transmucosal” delivery of a drug and the like is meant toencompass all forms of delivery across or through a mucosal membrane.

The term “bioadhesion” as used herein refers to the process of adhesionof the dosage form(s) to a biological surface, e.g., mucosal membranes.

“Controlled drug delivery” refers to release or administration of a drugfrom a given dosage form in a controlled fashion in order to achieve thedesired pharmacokinetic profile in vivo. An aspect of “controlled” drugdelivery is the ability to manipulate the formulation and/or dosage formin order to establish the desired kinetics of drug release.

“Sustained drug delivery” refers to release or administration of a drugfrom a source (e.g., a drug formulation) in a sustained fashion over aprotracted yet specific period of time, that may extend from severalminutes to a few hours, days, weeks or months. In various embodimentsthe term “sustained” will be used to refer to delivery of consistentand/or substantially constant levels of drug over a time period rangingfrom a few minutes to a day, with a profile characterized by the absenceof an immediate release phase, such as the one obtained from IVadministration.

The term “T_(max)” as used herein means the time point of maximumobserved plasma concentration.

The term “C_(max)” as used herein means the maximum observed plasmaconcentration.

The term “plasma t_(1/2)” as used herein means the observed “plasmahalf-life” and represents the time required for the drug plasmaconcentration to reach the 50% of its maximal value (C_(max)). Thisfacilitates determination of the mean duration of pharmacologicaleffects. In addition, it facilitates direct and meaningful comparisonsof the duration of different test articles after delivery via the sameor different routes.

The term “Optimal Therapeutic Targeting Ratio” or “OTTR” represents theaverage time that the drug is present at therapeutic levels, defined astime within which the drug plasma concentration is maintained above 50%of C_(max) normalized by the drug's elimination half-life multiplied bythe ratio of the C_(max) obtained in the dosage form of interest overthe C_(max) following IV administration of equivalent doses and it iscalculated by the formula:OTTR=(C ^(IV) _(max) /C _(max))×(Dose/Dose^(IV))(Time above 50% of C_(max))/(Terminal^(IV) elimination half-life of the drug).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of rutin, galangin and progalangin-1.

FIG. 2 illustrates galangin and various pro-galantins.

FIG. 3A illustrates the identification of Rutin in the AlphaLisa assayas shown in the scattergraph from screening of a small clinical library.FIG. 3B schematically illustrates the primary AlphaLisa assay used fordetection of the BACE cleavage of MBP-C125.

FIG. 4 shows the results of screening a family of Bioflavonoids in theMBP-C125 & P5-P5′ cleavage by BACE to identify ASBIs.

FIG. 5A shows inhibition of sAPPβ in SH-SY5Y by rutin & galangin. 5Bshows binding of galangin to APP fragments by surface plasmon resonance(SPR).

FIG. 6A shows that galangin inhibits β-CTF production. Chinese HamsterOvary (CHO) cells stably over-expressing human APP were treated with 10μM Galangin or DMSO for 24 hr, then the cell lysates and conditionedmedia were collected. The levels of full length APP and β-CTF in thecell lysates and the levels of sAPPα in the conditioned media weredetected by western blot. FIG. 6B shows that galangin does not inhibitBACE1 cleavage of neuregulin1. BACE1-dependent cleavage of SEAP-NRG1-β1was evaluated by SEAP assays with supernatants of control humanembryonic kidney 293 (HEK293) cells (SEAP-NRG1-β1) or HEK293 cellsco-expressing BACE1 (SEAP-NRG1-β1+BACE1), treated with DMSO or Galangin(10 μM). An increase in shedding of the SEAP-NRG1-b1 fusion protein wasmeasured upon BACE1 co-expression. Shedding in co-expressing cells wasnot suppressed by Galangin. Error bars indicate SEM. (N.S., p>0.05; n=4,student t test).

FIGS. 7A and 7B illustrate inhibition of APP-Gal4 and APLP2-Gal4transactivation by galangin. FIG. 7A: Potent transactivation oftranscription was achieved with APP fused to the Gal4 DNA binding domainwhen both Mint3 and TAZ were present. This transactivation was inhibitedby Galangin and BACE inhibitor IV. FIG. 7B: Potent transactivation oftranscription was also achieved with APLP2 fused to the Gal4 DNA bindingdomain when both Mint3 and TAZ were present. This transactivation wasinhibited by Galangin and BACE inhibitor IV. Diagrams exhibitexperiments in which cells were co-transfected with a Gal4-luciferasereporter plasmid (to measure transactivation), a β-galactosidase plasmid(to normalize for transfection efficiency), and the test plasmids(Mint3, TAZ and APP-Gal4 or APLP2-Gal4). The normalized luciferaseactivity is expressed as a percentage of the DMSO treated control (FIGS.7A, 7B). Error bars indicate SEM. (*, p<0.05; **, p<0.01; ***, p<0.001;n=3, student t test).

FIG. 8 illustrates the pharmacokinetics for galangin and progalangin(PG-1). Plasma levels were greater than brain tissue levels for bothgalangin and PG-1, but galangin levels were much higher after PG-1injection than galangin itself.

FIG. 9, panels A-D, show Aβ1-40 and Aβ1-42 levels. Aβ1-40 levels wereslightly lower in the brains of galangin-treated mice (panel A) andAβ1-42 was unchanged. Both Aβ1-40 and Aβ1-42 were slightly lowered inbrains of pro-galangin I-treated mice. Aβ1-40 (panel A) goes downslightly and Aβ1-42 (panel B) stays the same. Both Aβ1-40 (panel C) andAβ1-42 (panel D) go down with pro-galangin-1. For siblings only all N=3.

FIG. 10 illustrates a molecular-clamp model for ASBI activity.

FIG. 11 illustrates a synthesis scheme for progalangin (compound 2).

DETAILED DESCRIPTION

In certain embodiments bioflavonoid analogues are identified that thatinhibit β-secretase mediated APP processing by a novel mechanism. Inparticular, it is believed these molecules inhibit the BACE cleavage ofthe MBP-C125 APP substrate, resulting in the inhibition of theproduction of C99 but not the β-site peptide substrate (P5-P5′). Inaddition, various bioflavonoids and analogues thereof identified hereininhibit sAPPβ in neuroblastoma SHSY5Y cells. Further it was demonstratedthat the inhibitory activity is associated with binding to the MBP-C125substrate. Accordingly, these molecules appear to be APP specific BACEinhibitors (ASBIs) and provide a new mechanism to modulate APPprocessing. These ASBIs can be used in the treatment and/or prophylaxisof pathogolies characterized by pathological APP processing (e.g.,Alzheimer's disease, pre-Alzheimer's conditions such as MCI orpre-symptomatic MCI, and the like).

The ASBIs are at least selective and appear to be specific for the APPsubstrate and are believed to show fewer undesired side-effects becausethe ASBIs are typically not active on other substrates for the enzyme.With respect to inhibitors of γ-secretase, substrates other than APP,such as Notch, raise concerns for potential side effects of γ-secretaseinhibition, and the recent failure of the γ-secretase inhibitor,semagacestat, serves to reinforce such concerns. Similarly in the caseof BACE, for example, inhibition of non-APP substrates such as PSGL1 orLRP can produce adverse side-effects. Therefore, the optimal BACEinhibitor would be one that would bind not to BACE but rather to APP,leading to APP-specific BACE inhibition (ASBI).

Without being bound to a particular theory it is believed that suchASBIs would interact with APP or the APP-BACE complex (“inactive”complex) at the membrane and prevent its transition to the “active”complex in early endosomes, where at pH<5 BACE is fully active (see,e.g., FIG. 10). Some β-site binding antibodies have been shown to blockthe cleavage of APP by BACE and also work in animal models of AD,however for effective pharmaceutical development small organic moleculesare typically preferred to relatively large biomolecules such asantibodies.

The data described herein on the identification of the first ASBIs,demonstrates that such an approach is feasible.APP-Specific-BACE-Inhibitors (ASBIs) inhibit the BACE cleavage of theAmyloid Precursor Protein (APP) but not the proteolytic cleavage ofother substrates. Such therapeutics are believed to represent a newclass of Alzheimer's disease (or other amyloidogenic disease)therapeutics.

Initially a clinical library of 448 compounds was screened and thisassay led to the identification of a single bioflavonoid thatspecifically inhibited the MBP-C125 substrate of BACE while notpreventing the cleavage of the P5-P5′ substrate. This bioflavonoid Rutin(see, e.g., FIG. 1) is a nutritional supplement was also found toinhibit sAPPβ in cells. A panel of bioflavonoids was then tested in theASBI and sAPPβ assay in cell culture. This testing identified a secondbioflavonoid Galangin (see, e.g., FIG. 1) another nutritional supplementwith known human use that was effective in the ASBI assay and in cellsin preventing the BACE cleavage of APP. Galangin also has been reportedto be an inhibitor of acetylcholine esterase (AChE). Using a simplenitrocellulose filter ligand-binding assay initial binding of variousbioflavonoids to the MBP-C125 substrate was demonstrated. A panel ofbioflavonoids were screened in the ASBI assay. Rutin and Galangin wereidentified as effective in modulating sAPPβ levels in cells and showsbinding to the APP substrate (see Example 1).

It was demonstrated that the bioflavonoids could inhibit BACE cleavageof APP and APLP2. A HEK-293 assay transfected with APP or APLP2-Gal4 toobtain this data. This assay system is described by Orcholski et al.(2011) J. Alzheimers Dis., 23(4):689-699. Transactivation is achievedupon transfection with Mint3 and Taz. The ASBI was expected to inhibitonly the transactivation of APP-Gal4, not that of APLP2-Gal4. For theseexperiments cells were co-transfected with a Gal4-luciferase reporterplasmid (to measure transactivation), a beta-galactosidase plasmid (tonormalize for transfection efficiency), and the test plasmidsidentified. The normalized luciferase activity was expressed as foldinduction over transcription by APP-Gal4 alone (FIG. 7A), or as foldinduction over APLP2-Gal4 alone (FIG. 7B). Preliminary testing ofgalangin at 10 μM in this assay showed that it inhibits APP-Gal4 andAPLP2-Gal4.

Initial pharmacokinetic evaluation of these two bioflavonoids in brainuptake assay using NTg mice showed that Rutin does not have any brainlevels at 10 mpk after a sc dose, while Galangin did show significantbrain levels (40 ng/g at 1 h) thus enabling its evaluation forproof-of-concept studies in the transgenic (Tg) mouse model. Treatmentof the Tg mice were done by sc route at 100 mpk over 5 days. Galanginwas then evaluated for its effect on sAPPα, and Aβ40 and Aβ42 (seeExample 1). The reduction of Aβ levels was very encouraging in thisstudy. Further increase in brain levels of galangin was possible using aprodrug of galangin (see, e.g., FIG. 1).

The examples provided herein indicate that certain bioflavanoids havethe ability to bind APP and inhibit the BACE cleavage of APP and APLP2thus suggest that they are APP specific BACE inhibitors. These moleculesrepresent a new class of therapeutics for Alzheimer's disease that wouldbe devoid of the potential toxicity from direct inhibition of BACE.Galangin was also shown to be effective in reducing Aβ40 and Aβ42 in theAD mouse model.

While Rutin was not effective in the mouse model it is believed this isdue to difficulty passing the blood brain barrier. However, this doesnot mean that rutin and derivatives or analogues thereof are unsuitablefor use in the methods described herein. Numerous methods fortransporting a molecule through the blood brain barrier and/or forcircumventing the blood brain barrier are known to those of skill in theart.

Typically mechanisms for drug targeting in the brain involve goingeither “through” or “behind” the BBB. In various embodiments modalitiesfor drug delivery through the BBB entail its disruption by osmoticmeans; biochemically by the use of vasoactive substances such asbradykinin; or even by localized exposure to high-intensity focusedultrasound (HIFU) (see, e.g., McDannold et al. (2008) Ultrasound inMedicine and Biology, 34(5): 834-840). Other methods used to get throughthe BBB may entail the use of endogenous transport systems, includingcarrier-mediated transporters such as glucose and amino acid carriers;receptor-mediated transcytosis; and the blocking of active effluxtransporters such as p-glycoprotein. Methods for drug delivery behindthe BBB also include intracerebral implantation (such as with needles)and convection-enhanced distribution. In certain embodiments mMannitolcan be used in bypassing the BBB. Nanoparticles can also help in thetransfer of drugs across the BBB (see, e.g., Silva, (2008). BMCNeuroscience, 9:S4, and the like).

In view of the discovery of ASBI activity in Galangin and Rutin, similaractivity is believed to exist in a number of additional bioflavonoidanalogues described herein. Particular ASBI activity of any of theseanalogues can readily be further confirmed using, for example, theassays described above and illustrated in the Examples provided herein.

The sequential cleavage of APP by membrane-bound proteases β-secretaseand γ-secretase results in the formation of Aβ. The β-Site APP cleavageenzyme-1 (BACE1) was identified as the major β-secretase activity thatmediates the first cleavage of APP in the β-amyloidogenic pathway. Inview of the ability of the ASBI compounds described herein tospecifically block BACE1 activity at APP, it is believed (and the datapresented herein show) that these ASBI compounds can lower Aβ levels orprevent the formation of the neurotoxic Aβ species. Accordingly, thesecompounds are believed to prevent or slow the progression of the diseaseand/or to prevent or slow the progression of pre-clinical manifestationsof the amyloidogenic disease pathway.

Accordingly it is believed that these agents can be used to prevent ordelay the onset of a pre-Alzheimer's cognitive dysfunction, and/or toameliorate one or more symptoms of a pre-Alzheimer's cognitivedysfunction, and/or to prevent or delay the progression of apre-Alzheimer's condition or cognitive dysfunction to Alzheimer'sdisease, and/or to promote the processing of amyloid precursor protein(APP) by the non-amyloidogenic pathway. In certain embodiments theseagents can be used in the treatment of Alzheimer's disease (e.g., tolessen the severity of the disease, and/or to ameliorate one or moresymptoms of the disease, and/or to slow the progression of the disease).

Therapeutic and Prophylactic Methods.

In various embodiments therapeutic and/or prophylactic methods areprovided that utilize the active agent(s) (e.g., ASBIs such as galangin,rutin, and analogues, derivatives, or prodrugs thereof, or tautomer(s)or stereoisomer(s) thereof, or pharmaceutically acceptable salts orsolvates of said ASBI(s), said stereoisomer(s), or said tautomer(s), oranalogues, derivatives, or prodrugs thereof) are provided. Typically themethods involve administering one or more active agent(s) to a subject(e.g., to a human in need thereof) in an amount sufficient to realizethe desired therapeutic or prophylactic result.

Prophylaxis

In certain embodiments active agent(s) (e.g., ASBIs such as galangin,rutin, and analogues, derivatives, or prodrugs thereof, or tautomer(s)or stereoisomer(s) thereof, or pharmaceutically acceptable salts orsolvates of said ASBI(s), said stereoisomer(s), or said tautomer(s), oranalogues, derivatives, or prodrugs thereof) are utilized in variousprophylactic contexts. Thus, for example, ion certain embodiments, theactive agent(s) can be used to prevent or delay the onset of apre-Alzheimer's cognitive dysfunction, and/or to ameliorate one moresymptoms of a pre-Alzheimer's condition and/or cognitive dysfunction,and/or to prevent or delaying the progression of a pre-Alzheimer'scondition and/or cognitive dysfunction to Alzheimer's disease.

Accordingly in certain embodiments, the prophylactic methods describedherein are contemplated for subjects identified as “at risk” and/or ashaving evidence of early Alzheimer's Disease (AD) pathological changes,but who do not meet clinical criteria for MCI or dementia. Without beingbound to a particular theory, it is believed that even this“preclinical” stage of the disease represents a continuum fromcompletely asymptomatic individuals with biomarker evidence suggestiveof AD-pathophysiological process(es) (abbreviated as AD-P, see, e.g.,Sperling et. al. (2011) Alzheimer's & Dementia, 1-13) at risk forprogression to AD dementia to biomarker-positive individuals who arealready demonstrating very subtle decline but not yet meetingstandardized criteria for MCI (see, e.g., Albert et al. (2011)Alzheimer's and Dementia, 1-10 (doi:10.1016/j.jalz.2011.03.008).

This latter group of individuals might be classified as “Not normal, notMCI” but would be can be designated “pre-symptomatic” or “pre-clinicalor “asymptomatic” or “premanifest”). In various embodiments thiscontinuum of pre-symptomatic AD can also encompass (1) individuals whocarry one or more apolipoprotein E (APOE) ε4 alleles who are known orbelieved to have an increased risk of developing AD dementia, at thepoint they are AD-P biomarker-positive, and (2) carriers of autosomaldominant mutations, who are in the presymptomatic biomarker-positivestage of their illness, and who will almost certainly manifest clinicalsymptoms and progress to dementia.

A biomarker model has been proposed in which the most widely validatedbiomarkers of AD-P become abnormal and likewise reach a ceiling in anordered manner (see, e.g., Jack et al. (2010) Lancet Neurol., 9:119-128). This biomarker model parallels proposed pathophysiologicalsequence of (pre-AD/AD), and is relevant to tracking the preclinical(asymptomatic) stages of AD (see, e.g., FIG. 3 in Sperling et al. (2011)Alzheimer's & Dementia, 1-13). Biomarkers of brain amyloidosis include,but are not limited to reductions in CSF Aβ₄₂ and increased amyloidtracer retention on positron emission tomography (PET) imaging. ElevatedCSF tau is not specific to AD and is thought to be a biomarker ofneuronal injury. Decreased fluorodeoxyglucose 18F (FDG) uptake on PETwith a temporoparietal pattern of hypometabolism is a biomarker ofAD-related synaptic dysfunction. Brain atrophy on structural magneticresonance imaging (MRI) in a characteristic pattern involving the medialtemporal lobes, paralimbic and temporoparietal cortices is a biomarkerof AD-related neurodegeneration. Other markers include, but are notlimited to volumetric MRI, FDG-PET, or plasma biomarkers (see, e.g.,Vemuri et al. (2009) Neurology, 73: 294-301; Yaffe et al. (2011) JAMA305: 261-266).

In certain embodiments the subjects suitable for the prophylacticmethods contemplated herein include, but are not limited to subjectcharacterized as having asymptomatic asymptomatic cerebral amyloidosis.In various embodiments these individuals have biomarker evidence of Aβaccumulation with elevated tracer retention on PET amyloid imagingand/or low Aβ42 in CSF assay, but typically no detectable evidence ofadditional brain alterations suggestive of neurodegeneration or subtlecognitive and/or behavioral symptomatology.

It is noted that currently available CSF and PET imaging biomarkers ofAβ primarily provide evidence of amyloid accumulation and deposition offibrillar forms of amyloid. Data suggest that soluble or oligomericforms of Aβ are likely in equilibrium with plaques, which may serve asreservoirs. In certain embodiments it is contemplated that there is anidentifiable preplaque stage in which only soluble forms of Aβ arepresent. In certain embodiments it is contemplated that oligomeric formsof amyloid may be critical in the pathological cascade, and provideuseful markers. In addition, early synaptic changes may be presentbefore evidence of amyloid accumulation.

In certain embodiments the subjects suitable for the prophylacticmethods contemplated herein include, but are not limited to, subjectscharacterized as amyloid positive with evidence of synaptic dysfunctionand/or early neurodegeneration. In various embodiments these subjectshave evidence of amyloid positivity and presence of one or more markersof “downstream” AD-P-related neuronal injury. Illustrative, butnon-limiting markers of neuronal injury include, but are not limited to(1) elevated CSF tau or phospho-tau, (2) hypometabolism in an AD-likepattern (i.e., posterior cingulate, precuneus, and/or temporoparietalcortices) on FDG-PET, and (3) cortical thinning/gray matter loss in aspecific anatomic distribution (i.e., lateral and medial parietal,posterior cingulate, and lateral temporal cortices) and/or hippocampalatrophy on volumetric MRI. Other markers include, but are not limited tofMRI measures of default network connectivity. In certain embodimentsearly synaptic dysfunction, as assessed by functional imaging techniquessuch as FDG-PET and fMRI, can be detectable before volumetric loss.Without being bound to a particular theory, it is believed thatamyloid-positive individuals with evidence of early neurodegenerationmay be farther down the trajectory (i.e., in later stages of preclinical(asymptomatic) AD).

In certain embodiments the subjects suitable for the prophylacticmethods contemplated herein include, but are not limited to, subjectscharacterized as amyloid positive with evidence of neurodegeneration andsubtle cognitive decline. Without being bound to a particular theory, itis believed that those individuals with biomarker evidence of amyloidaccumulation, early neurodegeneration, and evidence of subtle cognitivedecline are in the last stage of preclinical (asymptomatic) AD, and areapproaching the border zone with clinical criteria for mild cognitiveimpairment (MCI). These individuals may demonstrate evidence of declinefrom their own baseline (particularly if proxies of cognitive reserveare taken into consideration), even if they still perform within the“normal” range on standard cognitive measures. Without being bound to aparticular theory, it is believed that more sensitive cognitivemeasures, particularly with challenging episodic memory measures, maydetect very subtle cognitive impairment in amyloid-positive individuals.In certain embodiments criteria include, but are not limited to,self-complaint of memory decline or other subtle neurobehavioralchanges.

As indicated above, subjects/patients amenable to prophylactic methodsdescribed herein include individuals at risk of disease (e.g., apathology characterized by amyloid plaque formation such as MCI) but notshowing symptoms, as well as subjects presently showing certain symptomsor markers. It is known that the risk of MCI and later Alzheimer'sdisease generally increases with age. Accordingly, in asymptomaticsubjects with no other known risk factors, in certain embodiments,prophylactic application is contemplated for subjects over 50 years ofage, or subjects over 55 years of age, or subjects over 60 years of age,or subjects over 65 years of age, or subjects over 70 years of age, orsubjects over 75 years of age, or subjects over 80 years of age, inparticular to prevent or slow the onset or ultimate severity of mildcognitive impairment (MCI), and/or to slow or prevent the progressionfrom MCI to early stage Alzheimer's disease (AD).

In certain embodiments, the methods described herein present methods areespecially useful for individuals who do have a known genetic risk ofAlzheimer's disease (or other amyloidogenic pathologies), whether theyare asymptomatic or showing symptoms of disease. Such individualsinclude those having relatives who have experienced MCI or AD (e.g., aparent, a grandparent, a sibling), and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include, for example, mutations in the APPgene, particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy(1997) Trends. Neurosci., 20: 154-159). Other markers of risk includemutations in the presenilin genes (PS1 and PS2), family history of AD,having the familial Alzheimer's disease (FAD) mutation, the APOE ε4allele, hypercholesterolemia or atherosclerosis. Further susceptibilitygenes for the development of Alzheimer's disease are reviewed, e.g., inSleegers, et al. (2010) Trends Genet. 26(2): 84-93.

In some embodiments, the subject is asymptomatic but has familial and/orgenetic risk factors for developing MCI or Alzheimer's disease. Inasymptomatic patients, treatment can begin at any age (e.g., 20, 30, 40,50 years of age). Usually, however, it is not necessary to begintreatment until a patient reaches at least about 40, 50, 60 or 70 yearsof age.

In some embodiments, the subject is exhibiting symptoms, for example, ofmild cognitive impairment (MCI) or Alzheimer's disease (AD). Individualspresently suffering from Alzheimer's disease can be recognized fromcharacteristic dementia, as well as the presence of risk factorsdescribed above. In addition, a number of diagnostic tests are availablefor identifying individuals who have AD. These include measurement ofCSF Tau, phospho-tau (pTau), Aβ42 levels and C-terminally cleaved APPfragment (APPneo). Elevated total-Tau (tTau), phospho-Tau (pTau),APPneo, soluble Aβ40, pTau/Aβ42 ratio and tTau/Aβ42 ratio, and decreasedAβ42 levels, Aβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα levels, sAPPα/sAPPβratio, sAPPα/Aβ40 ratio, and sAPPα/Aβ42 ratio signify the presence ofAD. In some embodiments, the subject or patient is diagnosed as havingMCI. Increased levels of neural thread protein (NTP) in urine and/orincreased levels of α2-macroglobulin (α2M) and/or complement factor H(CFH) in plasma are also biomarkers of MCI and/or AD (see, e.g., Anoopet al. (2010) Int. J. Alzheimer's Dis. 2010:606802).

In certain embodiments, subjects amenable to treatment may haveage-associated memory impairment (AAMI), or mild cognitive impairment(MCI). The methods described herein are particularly well-suited to theprophylaxis and/or treatment of MCI. In such instances, the methods candelay or prevent the onset of MCI, and or reduce one or more symptomscharacteristic of MCI and/or delay or prevent the progression from MCIto early-, mid- or late-stage Alzheimer's disease or reduce the ultimateseverity of the disease.

Mild Cognitive Impairment (MCI)

Mild cognitive impairment (MCI, also known as incipient dementia, orisolated memory impairment) is a diagnosis given to individuals who havecognitive impairments beyond that expected for their age and education,but that typically do not interfere significantly with their dailyactivities (see, e.g., Petersen et al. (1999) Arch. Neurol. 56(3):303-308). It is considered in many instances to be a boundary ortransitional stage between normal aging and dementia. Although MCI canpresent with a variety of symptoms, when memory loss is the predominantsymptom it is termed “amnestic MCI” and is frequently seen as a riskfactor for Alzheimer's disease (see, e.g., Grundman et al. (2004) Arch.Neurol. 61(1): 59-66; and on the internet aten.wikipedia.org/wiki/Mild_cognitive_impairment-cite_note-Grundman-1).When individuals have impairments in domains other than memory it isoften classified as non-amnestic single- or multiple-domain MCI andthese individuals are believed to be more likely to convert to otherdementias (e.g. dementia with Lewy bodies). There is evidence suggestingthat while amnestic MCI patients may not meet neuropathologic criteriafor Alzheimer's disease, patients may be in a transitional stage ofevolving Alzheimer's disease; patients in this hypothesized transitionalstage demonstrated diffuse amyloid in the neocortex and frequentneurofibrillary tangles in the medial temporal lobe (see, e.g., Petersenet al. (2006) Arch. Neurol. 63(5): 665-72).

The diagnosis of MCI typically involves a comprehensive clinicalassessment including clinical observation, neuroimaging, blood tests andneuropsychological testing. In certain embodiments diagnostic criteriafor MIC include, but are not limited to those described by Albert et al.(2011) Alzheimer's & Dementia. 1-10. As described therein, diagnosticcriteria include (1) core clinical criteria that could be used byhealthcare providers without access to advanced imaging techniques orcerebrospinal fluid analysis, and (2) research criteria that could beused in clinical research settings, including clinical trials. Thesecond set of criteria incorporate the use of biomarkers based onimaging and cerebrospinal fluid measures. The final set of criteria formild cognitive impairment due to AD has four levels of certainty,depending on the presence and nature of the biomarker findings.

In certain embodiments clinical evaluation/diagnosis of MCI involves:(1) Concern reflecting a change in cognition reported by patient orinformant or clinician (i.e., historical or observed evidence of declineover time); (2) Objective evidence of Impairment in one or morecognitive domains, typically including memory (i.e., formal or bedsidetesting to establish level of cognitive function in multiple domains);(3) Preservation of independence in functional abilities; (4) Notdemented; and in certain embodiments, (5) An etiology of MCI consistentwith AD pathophysiological processes. Typically vascular, traumatic,medical causes of cognitive decline, are ruled out where possible. Incertain embodiments, evidence of longitudinal decline in cognition isidentified, when feasible. Diagnosis is reinforced by a historyconsistent with AD genetic factors, where relevant.

With respect to impairment in cognitive domain(s), there should beevidence of concern about a change in cognition, in comparison with theperson's previous level. There should be evidence of lower performancein one or more cognitive domains that is greater than would be expectedfor the patient's age and educational background. If repeatedassessments are available, then a decline in performance should beevident over time. This change can occur in a variety of cognitivedomains, including memory, executive function, attention, language, andvisuospatial skills. An impairment in episodic memory (i.e., the abilityto learn and retain new information) is seen most commonly in MCIpatients who subsequently progress to a diagnosis of AD dementia.

With respect to preservation of independence in functional abilities, itis noted that persons with MCI commonly have mild problems performingcomplex functional tasks which they used to perform shopping. They maytake more time, be less efficient, and make more errors at performingsuch activities than in the past. Nevertheless, they generally maintaintheir independence of function in daily life, with minimal aids orassistance.

With respect to dementia, the cognitive changes should be sufficientlymild that there is no evidence of a significant impairment in social oroccupational functioning. If an individual has only been evaluated once,change will be inferred from the history and/or evidence that cognitiveperformance is impaired beyond what would have been expected for thatindividual.

Cognitive testing is optimal for objectively assessing the degree ofcognitive impairment for an individual. Scores on cognitive tests forindividuals with MCI are typically 1 to 1.5 standard deviations belowthe mean for their age and education matched peers on culturallyappropriate normative data (i.e., for the impaired domain(s), whenavailable).

Episodic memory (i.e., the ability to learn and retain new information)is most commonly seen in MCI patients who subsequently progress to adiagnosis of AD dementia. There are a variety of episodic memory teststhat are useful for identifying those MCI patients who have a highlikelihood of progressing to AD dementia within a few years. These teststypically assess both immediate and delayed recall, so that it ispossible to determine retention over a delay. Many, although not all, ofthe tests that have proven useful in this regard are wordlist learningtests with multiple trials. Such tests reveal the rate of learning overtime, as well as the maximum amount acquired over the course of thelearning trials. They are also useful for demonstrating that theindividual is, in fact, paying attention to the task on immediaterecall, which then can be used as a baseline to assess the relativeamount of material retained on delayed recall. Examples of such testsinclude (but are not limited to: the Free and Cued Selective RemindingTest, the Rey Auditory Verbal Learning Test, and the California VerbalLearning Test. Other episodic memory measures include, but are notlimited to: immediate and delayed recall of a paragraph such as theLogical Memory I and II of the Wechsler Memory Scale Revised (or otherversions) and immediate and delayed recall of nonverbal materials, suchas the Visual Reproduction subtests of the Wechsler Memory Scale-RevisedI and II.

Because other cognitive domains can be impaired among individuals withMCI, it is desirable to examine domains in addition to memory. Theseinclude, but are not limited to executive functions (e.g., set-shifting,reasoning, problem-solving, planning), language (e.g., naming, fluency,expressive speech, and comprehension), visuospatial skills, andattentional control (e.g., simple and divided attention). Many clinicalneuropsychological measures are available to assess these cognitivedomains, including (but not limited to the Trail Making Test (executivefunction), the Boston Naming Test, letter and category fluency(language), figure copying (spatial skills), and digit span forward(attention).

As indicated above, genetic factors can be incorporated into thediagnosis of MCI. If an autosomal dominant form of AD is known to bepresent (i.e., mutation in APP, PS1, PS2), then the development of MCIis most likely the predursor to AD dementia. The large majority of thesecases develop early onset AD (i.e., onset below 65 years of age).

In addition, there are genetic influences on the development of lateonset AD dementia. For example, the presence of one or two 84 alleles inthe apolipoprotein E (APOE) gene is a genetic variant broadly acceptedas increasing risk for late-onset AD dementia. Evidence suggests that anindividual who meets the clinical, cognitive, and etiologic criteria forMCI, and is also APOE ε4 positive, is more likely to progress to ADdementia within a few years than an individual without this geneticcharacteristic. It is believed that additional genes play an important,but smaller role than APOE and also confer changes in risk forprogression to AD dementia (see, e.g., Bertram et al. (2010) Neuron, 21:270-281).

In certain embodiments subjects suitable for the prophylactic methodsdescribed herein include, but need not be limited to subjects identifiedhaving one or more of the core clinical criteria described above and/orsubjects identified with one or more “research criteria” for MCI, e.g.,as described below.

“Research criteria” for the identification/prognosis of MCI include, butare not limited to biomarkers that increase the likelihood that MCIsyndrome is due to the pathophysiological processes of AD. Without beingbound to a particular theory, it is believed that the conjointapplication of clinical criteria and biomarkers can result in variouslevels of certainty that the MCI syndrome is due to ADpathophysiological processes. In certain embodiments, two categories ofbiomarkers have been the most studied and applied to clinical outcomesare contemplated. These include “Aβ” (which includes CSF Aβ₄₂ and/or PETamyloid imaging) and “biomarkers of neuronal injury” (which include, butare not limited to CSF tau/p-tau, hippocampal, or medial temporal lobeatrophy on MRI, and temporoparietal/precuneus hypometabolism orhypoperfusion on PET or SPECT).

Without being bound to a particular theory, it is believed that evidenceof both Aβ, and neuronal injury (either an increase in tau/p-tau orimaging biomarkers in a topographical pattern characteristic of AD),together confers the highest probability that the AD pathophysiologicalprocess is present. Conversely, if these biomarkers are negative, thismay provide information concerning the likelihood of an alternatediagnosis. It is recognized that biomarker findings may be contradictoryand accordingly any biomarker combination is indicative (an indicator)used on the context of a differential diagnosis and not itselfdispositive. It is recognized that varying severities of an abnormalitymay confer different likelihoods or prognoses, that are difficult toquantify accurately for broad application.

For those potential MCI subjects whose clinical and cognitive MCIsyndrome is consistent with AD as the etiology, the addition ofbiomarker analysis effects levels of certainty in the diagnosis. In themost typical example in which the clinical and cognitive syndrome of MCIhas been established, including evidence of an episodic memory disorderand a presumed degenerative etiology, the most likely cause is theneurodegenerative process of AD. However, the eventual outcome still hasvariable degrees of certainty. The likelihood of progression to ADdementia will vary with the severity of the cognitive decline and thenature of the evidence suggesting that AD pathophysiology is theunderlying cause. Without being bound to a particular theory it isbelieved that positive biomarkers reflecting neuronal injury increasethe likelihood that progression to dementia will occur within a fewyears and that positive findings reflecting both Ab accumulation andneuronal injury together confer the highest likelihood that thediagnosis is MCI due to AD.

A positive Aβ biomarker and a positive biomarker of neuronal injuryprovide an indication that the MCI syndrome is due to AD processes andthe subject is well suited for the methods described herein.

A positive Aβ biomarker in a situation in which neuronal injurybiomarkers have not been or cannot be tested or a positive biomarker ofneuronal injury in a situation in which Aβ biomarkers have not been orcannot be tested indicate an intermediate likelihood that the MCIsyndrome is due to AD. Such subjects are believed to be is well suitedfor the methods described herein

Negative biomarkers for both Aβ and neuronal injury suggest that the MCIsyndrome is not due to AD. In such instances the subjects may not bewell suited for the methods described herein.

There is evidence that magnetic resonance imaging can observedeterioration, including progressive loss of gray matter in the brain,from mild cognitive impairment to full-blown Alzheimer disease (see,e.g., Whitwell et al. (2008) Neurology 70(7): 512-520). A techniqueknown as PiB PET imaging is used to clearly show the sites and shapes ofbeta amyloid deposits in living subjects using a C11 tracer that bindsselectively to such deposits (see, e.g., Jack et al. (2008) Brain 131(Pt 3): 665-680).

In certain embodiments, MCI is typically diagnosed when there is 1)Evidence of memory impairment; 2) Preservation of general cognitive andfunctional abilities; and 3) Absence of diagnosed dementia.

In certain embodiments MCI and stages of Alzheimer's disease can beidentified/categorized, in part by Clinical Dementia Rating (CDR)scores. The CDR is a five point scale used to characterize six domainsof cognitive and functional performance applicable to Alzheimer diseaseand related dementias: Memory, Orientation, Judgment & Problem Solving,Community Affairs, Home & Hobbies, and Personal Care. The necessaryinformation to make each rating is obtained through a semi-structuredinterview of the patient and a reliable informant or collateral source(e.g., family member).

The CDR table provides descriptive anchors that guide the clinician inmaking appropriate ratings based on interview data and clinicaljudgment. In addition to ratings for each domain, an overall CDR scoremay be calculated through the use of an algorithm. This score is usefulfor characterizing and tracking a patient's level ofimpairment/dementia: 0=Normal; 0.5=Very Mild Dementia; 1=Mild Dementia;2=Moderate Dementia; and 3=Severe Dementia. An illustrative CDR table isshown in Table 1.

TABLE 1 Illustrative clinical dementia rating (CDR) table. Impairment:None Questionable Mild Moderate Severe CDR: 0 0.5 1 2 3 Memory No memoryConsistent Moderate Severe Severe loss or slight slight memory loss;memory memory inconsistent forgetfulness; more marked loss; only loss;only forgetfulness partial for recent highly fragments recollectionevents; defect learned remain of events' interferes material “benign”with retained; forgetfulness everyday new material activities rapidlylost Orientation Fully Fully Moderate Severe Oriented to orientedoriented difficulty difficulty person only except for with time withtime slight relationships; relationships; difficulty oriented forusually with time place at disoriented relationships examination; totime, often may have to place. geographic disorientation elsewhereJudgment & Solves Slight Moderate Severely Unable to Problem everydayimpairment difficulty in impaired in make Solving problems & in solvinghandling handling judgments handles problems, problems, problems, orsolve business & similarities, similarities similarities problemsfinancial and and and affairs well; differences differences;differences; judgment social social good in judgment judgment relationto usually usually past maintained impaired performance CommunityIndependent Slight Unable to No pretense of independent Affairs functionat impairment function function outside of home usual level in theseindependently Appears well Appears too in job, activities at theseenough to be ill to be shopping, activities taken to taken to volunteer,although may functions functions and social still be outside a outside agroups engaged in family home family some; home. appears normal tocasual inspection Home and Life at Life at home, Mild bit Only simple NoHobbies home, hobbies, and definite chores significant hobbies, andintellectual impairment preserved; function in intellectual interests offunction at very home interests slightly home; more restricted wellimpaired difficult interests, maintained chores poorly abandoned;maintained more complicated hobbies and interests abandoned PersonalFully capable of self-care Needs Requires Requires Care promptingassistance in much help dressing, with hygiene, personal keeping ofcare; personal frequent effects incontinence

A CDR rating of ˜0.5 or ˜0.5 to 1.0 is often considered clinicallyrelevant MCI. Higher CDR ratings can be indicative of progression intoAlzheimer's disease.

In certain embodiments administration of one or more agents describedherein (e.g., ASBIs such as galangin, rutin, and analogues, derivatives,or prodrugs thereof, or tautomer(s) or stereoisomer(s) thereof, orpharmaceutically acceptable salts or solvates of said ASBI(s), saidstereoisomer(s), or said tautomer(s), or analogues, derivatives, orprodrugs thereof) is deemed effective when there is a reduction in theCSF of levels of one or more components selected from the groupconsisting of Tau, phospho-Tau (pTau), APPneo, soluble Aβ40, solubleAβ42, and/or Aβ42/Aβ40 ratio, and/or when there is a reduction of theplaque load in the brain of the subject, and/or when there is areduction in the rate of plaque formation in the brain of the subject,and/or when there is an improvement in the cognitive abilities of thesubject, and/or when there is a perceived improvement in quality of lifeby the subject, and/or when there is a significant reduction in clinicaldementia rating (CDR), and/or when the rate of increase in clinicaldementia rating is slowed or stopped and/or when the progression fromMCI to early stage AD is slowed or stopped.

In some embodiments, a diagnosis of MCI can be determined by consideringthe results of several clinical tests. For example, Grundman, et al.,Arch Neurol (2004) 61:59-66, report that a diagnosis of MCI can beestablished with clinical efficiency using a simple memory test(paragraph recall) to establish an objective memory deficit, a measureof general cognition (Mini-Mental State Exam (MMSE), discussed ingreater detail below) to exclude a broader cognitive decline beyondmemory, and a structured clinical interview (CDR) with patients andcaregivers to verify the patient's memory complaint and memory loss andto ensure that the patient was not demented. Patients with MCI perform,on average, less than 1 standard deviation (SD) below normal onnonmemorycognitive measures included in the battery. Tests of learning,attention, perceptual speed, category fluency, and executive functionmay be impaired in patients with MCI, but these are far less prominentthan the memory deficit.

Alzheimer's Disease (AD).

In certain embodiments the active agent(s) (e.g., ASBIs such asgalangin, rutin, and analogues, derivatives, or prodrugs thereof, ortautomer(s) or stereoisomer(s) thereof, or pharmaceutically acceptablesalts or solvates of said ASBI(s), said stereoisomer(s), or saidtautomer(s), or analogues, derivatives, or prodrugs thereof) and/orformulations thereof are contemplated for the treatment of Alzheimer'sdisease. In such instances the methods described herein are useful inpreventing or slowing the onset of Alzheimer's disease (AD), in reducingthe severity of AD when the subject has transitioned to clinical ADdiagnosis, and/or in mitigating one or more symptoms of Alzheimer'sdisease.

In particular, where the Alzheimer's disease is early stage, the methodscan reduce or eliminate one or more symptoms characteristic of AD and/ordelay or prevent the progression from MCI to early or later stageAlzheimer's disease.

Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. Individuals presentlysuffering from Alzheimer's disease can be recognized from characteristicdementia, as well as the presence of risk factors described above. Inaddition, a number of diagnostic tests are available for identifyingindividuals who have AD. These include measurement of CSF Tau,phospho-tau (pTau), sAPPα, sAPPβ, Aβ40, Aβ42 levels and/or C terminallycleaved APP fragment (APPneo). Elevated Tau, pTau, sAPPβ and/or APPneo,and/or decreased sAPPα, soluble Aβ40 and/or soluble Aβ42 levels,particularly in the context of a differential diagnosis, can signify thepresence of AD.

In certain embodiments subjects amenable to treatment may haveAlzheimer's disease. Individuals suffering from Alzheimer's disease canalso be diagnosed by Alzheimer's disease and Related DisordersAssociation (ADRDA) criteria. The NINCDS-ADRDA Alzheimer's Criteria wereproposed in 1984 by the National Institute of Neurological andCommunicative Disorders and Stroke and the Alzheimer's Disease andRelated Disorders Association (now known as the Alzheimer's Association)and are among the most used in the diagnosis of Alzheimer's disease(AD). McKhann, et al. (1984) Neurology 34(7): 939-44. According to thesecriteria, the presence of cognitive impairment and a suspected dementiasyndrome should be confirmed by neuropsychological testing for aclinical diagnosis of possible or probable AD. However, histopathologicconfirmation (microscopic examination of brain tissue) is generally usedfor a dispositive diagnosis. The NINCDS-ADRDA Alzheimer's Criteriaspecify eight cognitive domains that may be impaired in AD: memory,language, perceptual skills, attention, constructive abilities,orientation, problem solving and functional abilities). These criteriahave shown good reliability and validity.

Baseline evaluations of patient function can made using classicpsychometric measures, such as the Mini-Mental State Exam (MMSE)(Folstein et al. (1975) J. Psychiatric Research 12 (3): 189-198), andthe Alzheimer's Disease Assessment Scale (ADAS), which is acomprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function (see, e.g., Rosen, et al. (1984) Am. J. Psychiatr.,141: 1356-1364). These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. The extent of diseaseprogression can be determined using a Mini-Mental State Exam (MMSE)(see, e.g., Folstein, et al. supra). Any score greater than or equal to25 points (out of 30) is effectively normal (intact). Below this, scorescan indicate severe (≤9 points), moderate (10-20 points) or mild (21-24points) Alzheimer's disease.

Alzheimer's disease can be broken down into various stages including: 1)Moderate cognitive decline (Mild or early-stage Alzheimer's disease), 2)Moderately severe cognitive decline (Moderate or mid-stage Alzheimer'sdisease), 3) Severe cognitive decline (Moderately severe or mid-stageAlzheimer's disease), and 4) Very severe cognitive decline (Severe orlate-stage Alzheimer's disease) as shown in Table 2.

TABLE 2 Illustrative stages of Alzheimer's disease. Moderate CognitiveDecline (Mild or early stage AD) At this stage, a careful medicalinterview detects clear-cut deficiencies in the following areas:Decreased knowledge of recent events. Impaired ability to performchallenging mental arithmetic. For example, to count backward from 100by 7 s. Decreased capacity to perform complex tasks, such as marketing,planning dinner for guests, or paying bills and managing finances.Reduced memory of personal history. The affected individual may seemsubdued and withdrawn, especially in socially or mentally challengingsituations. Moderately severe cognitive decline (Moderate or mid-stageAlzheimer's disease) Major gaps in memory and deficits in cognitivefunction emerge. Some assistance with day-to-day activities becomesessential. At this stage, individuals may: Be unable during a medicalinterview to recall such important details as their current address,their telephone number, or the name of the college or high school fromwhich they graduated. Become confused about where they are or about thedate, day of the week or season. Have trouble with less challengingmental arithmetic; for example, counting backward from 40 by 4 s or from20 by 2 s. Need help choosing proper clothing for the season or theoccasion. Usually retain substantial knowledge about themselves and knowtheir own name and the names of their spouse or children. Usuallyrequire no assistance with eating or using the toilet. Severe cognitivedecline (Moderately severe or mid-stage Alzheimer's disease) Memorydifficulties continue to worsen, significant personality changes mayemerge, and affected individuals need extensive help with dailyactivities. At this stage, individuals may: Lose most awareness ofrecent experiences and events as well as of their surroundings.Recollect their personal history imperfectly, although they generallyrecall their own name. Occasionally forget the name of their spouse orprimary caregiver but generally can distinguish familiar from unfamiliarfaces. Need help getting dressed properly; without supervision, may makesuch errors as putting pajamas over daytime clothes or shoes on wrongfeet. Experience disruption of their normal sleep/waking cycle. Needhelp with handling details of toileting (flushing toilet, wiping anddisposing of tissue properly). Have increasing episodes of urinary orfecal incontinence. Experience significant personality changes andbehavioral symptoms, including suspiciousness and delusions (forexample, believing that their caregiver is an impostor); hallucinations(seeing or hearing things that are not really there); or compulsive,repetitive behaviors such as hand-wringing or tissue shredding. Tend towander and become lost. Very severe cognitive decline (Severe orlate-stage Alzheimer's disease) This is the final stage of the diseasewhen individuals lose the ability to respond to their environment, theability to speak, and, ultimately, the ability to control movement.Frequently individuals lose their capacity for recognizable speech,although words or phrases may occasionally be uttered. Individuals needhelp with eating and toileting and there is general incontinence.Individuals lose the ability to walk without assistance, then theability to sit without support, the ability to smile, and the ability tohold their head up. Reflexes become abnormal and muscles grow rigid.Swallowing is impaired.

In various embodiments administration of one or more agents describedherein to subjects diagnosed with Alzheimer's disease is deemedeffective when the there is a reduction in the CSF of levels of one ormore components selected from the group consisting of Tau, phospho-Tau(pTau), APPneo, soluble Aβ40, soluble Aβ42, and/or and Aβ42/Aβ40 ratio,and/or when there is a reduction of the plaque load in the brain of thesubject, and/or when there is a reduction in the rate of plaqueformation in the brain of the subject, and/or when there is animprovement in the cognitive abilities of the subject, and/or when thereis a perceived improvement in quality of life by the subject, and/orwhen there is a significant reduction in clinical dementia rating (CDR)of the subject, and/or when the rate of increase in clinical dementiarating is slowed or stopped and/or when the progression of AD is slowedor stopped (e.g., when the transition from one stage to another aslisted in Table 3 is slowed or stopped).

In certain embodiments Subjects amenable to the present methodsgenerally are free of a neurological disease or disorder other thanAlzheimer's disease. For example, in certain embodiments, the subjectdoes not have and is not at risk of developing a neurological disease ordisorder such as Parkinson's disease, and/or schizophrenia, and/orpsychosis.

Active Agent(s).

The methods described herein are based, in part, on the discovery thatadministration of one or more active agents e.g., ASBIs such asgalangin, rutin, and analogues, derivatives, or prodrugs thereof finduse in the treatment and/or prophylaxis of diseases characterized byamyloid deposits in the brain, for example, mild cognitive impairment,Alzheimer's disease, macular degeneration, and the like.

Bioflavanoids.

In certain embodiments the active agent(s) used in the methods describedherein comprise a flavanoid such as galangin or rutin or a derivativeand/or analogue thereof. In certain embodiments the flavonoid ischaracterized by Formula I:

where R¹ is selected from the group consisting of OH, O-saccaharide,O-alkyl, O-trifluoromethyl, O-aryl, O-heteroaryl; R⁴ and R⁵ areindependently selected from the group consisting of H, OH, NH₂, O-alkyl,O-trifluoromethyl, S-alkyl, S-aryl, carboxylate, halogen, NH-alkyl,N,N-dialkyl, NHCO-alkyl, and heteroaryl, alkyl urea, and carbamate; andR² and R³ are independently selected from the group consisting of H, OH,NH₂, O-alkyl, O-trifluoromethyl, S-alkyl, S-aryl, carboxylate, halogen,NH-alkyl, N,N-dialkyl, NHCO-alkyl, heteroaryl, alkyl urea, andcarbamate.

In certain embodiments R² and/or R³ is OH. In certain embodiments R² isOH and R³ is OH. In certain embodiments R² and/or R³ are independentlyselected from the group consisting of O-alkyl, S-alkyl, NH-alkyl andNHCO-alkyl. In certain embodiments the alkyl component of the O-alkyl,S-alkyl, NH-alkyl and NHCO-alkyl is a C₁₋₁₂ alkyl, or a C₁₋₉ alkyl, or aC₁₋₆ alkyl, or a C₁₋₃ alkyl. In certain embodiments R² and/or R³ ishalogen. In certain embodiments R² is halogen and R³ is halogen. Incertain embodiments R² and/or R³ are independently selected from thegroup consisting of Cl, Br, Fl, and I. In certain embodiments R² and/orR³ is selected from the group consisting of S-aryl and heteroaryl. Incertain embodiments R² and R³ are independently selected S-aryl. Incertain embodiments R² and R³ are independently selected heteroaryl. Incertain embodiments R⁴ and/or R⁵ is OH. In certain embodiments R⁴ is Hand R⁵ is OH or R⁴ is OH and R⁵ is H. In certain embodiments R⁴ is OHand R⁵ is OH. In certain embodiments R⁴ and/or R⁵ is H. In certainembodiments R⁴ is H and R⁵ is H. In certain embodiments when R⁴ and/orR⁵ is OH, R¹ is O-Saccharide. In certain embodiments R⁴ and/or R⁵ areindependently selected from the group consisting of O-alkyl, S-alkyl,NH-alkyl and NHCO-alkyl. In certain embodiments R⁴ and R⁵ areindependently selected from the group consisting of O-alkyl, S-alkyl,NH-alkyl and NHCO-alkyl. In certain embodiments the alkyl component ofsaid O-alkyl, S-alkyl, NH-alkyl and NHCO-alkyl is a C₁₋₁₂ alkyl, or aC₁₋₉ alkyl, or a C₁₋₆ alkyl, or a C₁₋₃ alkyl. In certain embodiments R⁴and/or R⁵ is halogen. In certain embodiments R⁴ is halogen and R⁵ ishalogen. In certain embodiments R⁴ and/or R⁵ are independently selectedfrom the group consisting of Cl, Br, Fl, and I. In certain embodimentsR⁴ and/or R⁵ is selected from the group consisting of S-aryl and heterIn certain embodiments

R⁴ and R⁵ are independently selected heteroaryl. In certain embodimentsR¹ is O-Saccharide (e.g., O-monosaccharide, O-disaccharide,O-trisaccharide, etc.). In certain embodiments R¹ is O-alkyl,O-trifluoromethyl, O-aryl, or O-heteroaryl.

In certain embodiments the APP specific BACE inhibitor is galangin or aderivative thereof. In certain embodiments APP specific BACE inhibitoris rutin or a derivative thereof.

Methods of making galangin and/or rutin and/or the various derivativesthereof contemplated herein are known to those of skill in the art. Bothgalangin and rutin are commercially available as are certainderivatives.

These compounds can be further functionalized to prepare the variousderivatives and analogues described herein using methods well known tothose of skill in the art. For example the procedure described inexample 2 would be used in the synthesis of analogs using variouscommercially available Dihydroxy-2-phenyl-4H-chromen-4-ones. Thecobversion to the acetoxy groups would be done as described in example2. Treatment with dimethyldioxirane would used to convert to the3-hydroxyflavone. Further hydrolysis with mild base can be done toremove the acetoxy protecting groups, the crude mixture of the flavonederivatives can be purified by flash chromatography andrecrystallisation to obtain the desired flavone analogs.

Flavanoid Prodrugs.

In certain embodiments it is contemplated that the various flavonoidsdescribed herein can be provided as flavonoid prodrugs. Illustrativegalangin prodrugs are shown in FIG. 2.

In certain embodiments the prodrug is a galangin prodrug ischaracterized by Formula II:

where R¹, R², and R³ are H, or a protecting group that is removed invivo in a mammal, wherein at least one of R¹, R², and R³ is not H; andwherein said prodrug partially or completely inhibits BACE processing ofAPP when administered to a mammal.

In certain embodiments at least one of R¹, R², and R³ are independentlyselected from the group consisting of least one of R¹, R², and R³ areindependently selected from the group consisting of

In certain embodiments R¹ is H. In certain embodiments R² is Group a,above and R³ is Group a, b, c, d, or e, above. In certain embodiments R³is Group a, above and R² is Group a, b, c, d, or e, above. In certainembodiments, R² and R³ are both group a above.

In certain embodiments R¹ is H. In certain embodiments R² is Group b,above and R³ is Group a, b, c, d, or e, above. In certain embodiments R³is Group b, above and R² is Group a, b, c, d, or e, above. In certainembodiments, R² and R³ are both group b above.

In certain embodiments R¹ is H. In certain embodiments R² is Group c,above and R³ is Group a, b, c, d, or e, above. In certain embodiments R³is Group c, above and R² is Group a, b, c, d, or e, above. In certainembodiments, R² and R³ are both group c above.

In certain embodiments R¹ is H. In certain embodiments R² is Group d,above and R³ is Group a, b, c, d, or e, above. In certain embodiments R³is Group d, above and R² is Group a, b, c, d, or e, above. In certainembodiments, R² and R³ are both group d above.

In certain embodiments R¹ is H. In certain embodiments R² is Group e,above and R³ is Group a, b, c, d, or e, above. In certain embodiments R³is Group e, above and R² is Group a, b, c, d, or e, above. In certainembodiments, R² and R³ are both group e above.

Methods of preparing galangin prodrugs such as are described herein areknown to those of skill in the art.

One such protocol is illustrated in Example 2 (see synthesis scheme inFIG. 11 for the synthesis of compound 2.

The various active agents and synthesis schemes are intended to beillustrative and not limiting. Using the teachings provided herein,numerous other flavonoid, flavonoid derivative, and flavonoid prodrugASBI compounds can be synthesized and identified by one of skill in theart.

Pharmaceutical Formulations.

In certain embodiments one or more active agents described herein (e.g.,ASBIs such as galangin, rutin, and analogues, derivatives, or prodrugsthereof, or tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said ASBI(s), said stereoisomer(s), orsaid tautomer(s), or analogues, derivatives, or prodrugs thereof) areadministered to a mammal in need thereof, e.g., to a mammal at risk foror suffering from a pathology characterized by abnormal processing ofamyloid precursor proteins, a mammal at risk for progression of MCI toAlzheimer's disease, and so forth. In certain embodiments the activeagent(s) are administered to prevent or delay the onset of apre-Alzheimer's condition and/or cognitive dysfunction, and/or toameliorate one or more symptoms of a pre-Alzheimer's cognitivedysfunction, and/or to prevent or delay the progression of apre-Alzheimer's condition or cognitive dysfunction to Alzheimer'sdisease, and/or to promote the processing of amyloid precursor protein(APP) by a non-amyloidogenic pathway.

The active agent(s) can be administered in the “native” form or, ifdesired, in the form of salts, esters, amides, prodrugs, derivatives,and the like, provided the salt, ester, amide, prodrug or derivative issuitable pharmacologically, i.e., effective in the present method(s).Salts, esters, amides, prodrugs and other derivatives of the activeagents can be prepared using standard procedures known to those skilledin the art of synthetic organic chemistry and described, for example, byMarch (1992) Advanced Organic Chemistry; Reactions, Mechanisms andStructure, 4th Ed. N.Y. Wiley-Interscience, and as described above.

For example, a pharmaceutically acceptable salt can be prepared for anyof the agent(s) described herein having a functionality capable offorming a salt. A pharmaceutically acceptable salt is any salt thatretains the activity of the parent compound and does not impart anydeleterious or untoward effect on the subject to which it isadministered and in the context in which it is administered.

In various embodiments pharmaceutically acceptable salts may be derivedfrom organic or inorganic bases. The salt may be a mono or polyvalention. Of particular interest are the inorganic ions, lithium, sodium,potassium, calcium, and magnesium. Organic salts may be made withamines, particularly ammonium salts such as mono-, di- and trialkylamines or ethanol amines. Salts may also be formed with caffeine,tromethamine and similar molecules.

Methods of formulating pharmaceutically active agents as salts, esters,amide, prodrugs, and the like are well known to those of skill in theart. For example, salts can be prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include, but are not limited to bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like, as well asinorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. An acid addition saltcan be reconverted to the free base by treatment with a suitable base.Certain particularly preferred acid addition salts of the active agentsherein include halide salts, such as may be prepared using hydrochloricor hydrobromic acids. Conversely, preparation of basic salts of theactive agents of this invention are prepared in a similar manner using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or thelike. Particularly preferred basic salts include alkali metal salts,e.g., the sodium salt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH units lower than the pKa ofthe drug. Similarly, for the preparation of salt forms of acidic drugs,the pKa of the counterion is preferably at least about 2 pH units higherthan the pKa of the drug. This permits the counterion to bring thesolution's pH to a level lower than the pH_(max) to reach the saltplateau, at which the solubility of salt prevails over the solubility offree acid or base. The generalized rule of difference in pKa units ofthe ionizable group in the active pharmaceutical ingredient (API) and inthe acid or base is meant to make the proton transfer energeticallyfavorable. When the pKa of the API and counterion are not significantlydifferent, a solid complex may form but may rapidly disproportionate(i.e., break down into the individual entities of drug and counterion)in an aqueous environment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups that are present within the molecular structureof the active agent. In certain embodiments, the esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moieties thatare derived from carboxylic acids of the formula RCOOH where R is alky,and preferably is lower alkyl. Esters can be reconverted to the freeacids, if desired, by using conventional hydrogenolysis or hydrolysisprocedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

In various embodiments, the active agents identified herein (e.g., ASBIssuch as galangin, rutin, and analogues, derivatives, or prodrugsthereof, or tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said ASBI(s), said stereoisomer(s), orsaid tautomer(s), or analogues, derivatives, or prodrugs thereof) areuseful for parenteral administration, topical administration, oraladministration, nasal administration (or otherwise inhaled), rectaladministration, or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment of one ormore of the pathologies/indications described herein (e.g., pathologiescharacterized by excess amyloid plaque formation and/or deposition orundesired amyloid or pre-amyloid processing).

The active agents described herein can also be combined with apharmaceutically acceptable carrier (excipient) to form apharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds, particularly of use in thepreparation of tablets, capsules, gel caps, and the like include, butare not limited to binders, diluent/fillers, disentegrants, lubricants,suspending agents, and the like.

In certain embodiments, to manufacture an oral dosage form (e.g., atablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.),an optional disintegrator (e.g. calcium carbonate,carboxymethylcellulose calcium, sodium starch glycollate, crospovidoneetc.), a binder (e.g. alpha-starch, gum arabic, microcrystallinecellulose, carboxymethylcellulose, polyvinylpyrrolidone,hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant(e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), forinstance, are added to the active component or components (e.g., ASBIssuch as galangin, rutin, and analogues, derivatives, or prodrugsthereof, or tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said ASBI(s), said stereoisomer(s), orsaid tautomer(s), or analogues, derivatives, or prodrugs thereof) andthe resulting composition is compressed. Where necessary the compressedproduct is coated, e.g., using known methods for masking the taste orfor enteric dissolution or sustained release. Suitable coating materialsinclude, but are not limited to ethyl-cellulose, hydroxymethylcellulose,POLYOX®yethylene glycol, cellulose acetate phthalate,hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas,Germany; methacrylic-acrylic copolymer).

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

In certain embodiments the excipients are sterile and generally free ofundesirable matter. These compositions can be sterilized byconventional, well-known sterilization techniques. For various oraldosage form excipients such as tablets and capsules sterility is notrequired. The USP/NF standard is usually sufficient.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Suitable unitdosage forms, include, but are not limited to powders, tablets, pills,capsules, lozenges, suppositories, patches, nasal sprays, injectables,implantable sustained-release formulations, mucoadherent films, topicalvarnishes, lipid complexes, etc.

Pharmaceutical compositions comprising the active agents describedherein (e.g., ASBIs such as galangin, rutin, and analogues, derivatives,or prodrugs thereof, or tautomer(s) or stereoisomer(s) thereof, orpharmaceutically acceptable salts or solvates of said ASBI(s), saidstereoisomer(s), or said tautomer(s), or analogues, derivatives, orprodrugs thereof) can be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions can be formulated in a conventional manner using one ormore physiologically acceptable carriers, diluents, excipients orauxiliaries that facilitate processing of the active agent(s) intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

In certain embodiments, the active agents described herein areformulated for oral administration. For oral administration, suitableformulations can be readily formulated by combining the active agent(s)with pharmaceutically acceptable carriers suitable for oral deliverywell known in the art. Such carriers enable the active agent(s)described herein to be formulated as tablets, pills, dragees, caplets,lizenges, gelcaps, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients can include fillers such as sugars (e.g.,lactose, sucrose, mannitol and sorbitol), cellulose preparations (e.g.,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose), synthetic poymers (e.g., polyvinylpyrrolidone(PVP)), granulating agents; and binding agents. If desired,disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques. The preparation ofenteric-coated particles is disclosed for example in U.S. Pat. Nos.4,786,505 and 4,853,230.

For administration by inhalation, the active agent(s) are convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

In various embodiments the active agent(s) can be formulated in rectalor vaginal compositions such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides. Methods of formulating active agents for rectal or vaginaldelivery are well known to those of skill in the art (see, e.g., Allen(2007) Suppositories, Pharmaceutical Press) and typically involvecombining the active agents with a suitable base (e.g., hydrophilic(PEG), lipophilic materials suc as cocoa butter or Witepsol W45),amphiphilic materials such as Suppocire AP and polyglycolized glyceride,and the like). The base is selected and compounded for a desiredmelting/delivery profile.

For topical administration the active agent(s) described herein (e.g.,ASBIs such as galangin, rutin, and analogues, derivatives, or prodrugsthereof, or tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said ASBI(s), said stereoisomer(s), orsaid tautomer(s), or analogues, derivatives, or prodrugs thereof) can beformulated as solutions, gels, ointments, creams, suspensions, and thelike as are well-known in the art.

In certain embodiments the active agents described herein are formulatedfor systemic administration (e.g., as an injectable) in accordance withstandard methods well known to those of skill in the art. Systemicformulations include, but are not limited to, those designed foradministration by injection, e.g. subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal oral or pulmonaryadministration. For injection, the active agents described herein can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiological saline buffer and/or in certain emulsion formulations. Thesolution(s) can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. In certain embodiments the activeagent(s) can be provided in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. For transmucosaladministration, and/or for blood/brain barrier passage, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art. Injectableformulations and inhalable formulations are generally provided as asterile or substantially sterile formulation.

In addition to the formulations described previously, the activeagent(s) may also be formulated as a depot preparations. Such longacting formulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the active agent(s) may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments the active agent(s) described herein can also bedelivered through the skin using conventional transdermal drug deliverysystems, i.e., transdermal “patches” wherein the active agent(s) aretypically contained within a laminated structure that serves as a drugdelivery device to be affixed to the skin. In such a structure, the drugcomposition is typically contained in a layer, or “reservoir,”underlying an upper backing layer. It will be appreciated that the term“reservoir” in this context refers to a quantity of “activeingredient(s)” that is ultimately available for delivery to the surfaceof the skin. Thus, for example, the “reservoir” may include the activeingredient(s) in an adhesive on a backing layer of the patch, or in anyof a variety of different matrix formulations known to those of skill inthe art. The patch may contain a single reservoir, or it may containmultiple reservoirs.

In one illustrative embodiment, the reservoir comprises a polymericmatrix of a pharmaceutically acceptable contact adhesive material thatserves to affix the system to the skin during drug delivery. Examples ofsuitable skin contact adhesive materials include, but are not limitedto, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

Alternatively, other pharmaceutical delivery systems can be employed.For example, liposomes, emulsions, and microemulsions/nanoemulsions arewell known examples of delivery vehicles that may be used to protect anddeliver pharmaceutically active compounds. Certain organic solvents suchas dimethylsulfoxide also can be employed, although usually at the costof greater toxicity.

In certain embodiments the active agent(s) described herein (e.g., ASBIssuch as galangin, rutin, and analogues, derivatives, or prodrugsthereof, or tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said ASBI(s), said stereoisomer(s), orsaid tautomer(s), or analogues, derivatives, or prodrugs thereof) areformulated in a nanoemulsion. Nanoemulsions include, but are not limitedto oil in water (O/W) nanoemulsions, and water in oil (W/O)nanoemulsions. Nanoemulsions can be defined as emulsions with meandroplet diameters ranging from about 20 to about 1000 nm. Usually, theaverage droplet size is between about 20 nm or 50 nm and about 500 nm.The terms sub-micron emulsion (SME) and mini-emulsion are used assynonyms.

Illustrative oil in water (O/W) nanoemulsions include, but are notlimited to: Surfactant micelles—micelles composed of small moleculessurfactants or detergents (e.g., SDS/PBS/2-propanol); Polymermicelles—micelles composed of polymer, copolymer, or block copolymersurfactants (e.g., Pluronic L64/PBS/2-propanol); Blendedmicelles—micelles in which there is more than one surfactant componentor in which one of the liquid phases (generally an alcohol or fatty acidcompound) participates in the formation of the micelle (e.g., octanoicacid/PBS/EtOH); Integral micelles—blended micelles in which the activeagent(s) serve as an auxiliary surfactant, forming an integral part ofthe micelle; and Pickering (solid phase) emulsions—emulsions in whichthe active agent(s) are associated with the exterior of a solidnanoparticle (e.g., polystyrene nanoparticles/PBS/no oil phase).

Illustrative water in oil (W/O) nanoemulsions include, but are notlimited to: Surfactant micelles—micelles composed of small moleculessurfactants or detergents (e.g., dioctyl sulfosuccinate/PBS/2-propanol,isopropylmyristate/PBS/2-propanol, etc.); Polymer micelles—micellescomposed of polymer, copolymer, or block copolymer surfactants (e.g.,PLURONIC® L121/PBS/2-propanol); Blended micelles—micelles in which thereis more than one surfactant component or in which one of the liquidphases (generally an alcohol or fatty acid compound) participates in theformation of the micelle (e.g., capric/caprylic diglyceride/PBS/EtOH);Integral micelles—blended micelles in which the active agent(s) serve asan auxiliary surfactant, forming an integral part of the micelle (e.g.,active agent/PBS/polypropylene glycol); and Pickering (solid phase)emulsions—emulsions in which the active agent(s) are associated with theexterior of a solid nanoparticle (e.g., chitosan nanoparticles/noaqueous phase/mineral oil).

As indicated above, in certain embodiments the nanoemulsions compriseone or more surfactants or detergents. In some embodiments thesurfactant is a non-anionic detergent (e.g., a polysorbate surfactant, apolyoxyethylene ether, etc.). Surfactants that find use in the presentinvention include, but are not limited to surfactants such as theTWEEN®, TRITON®, and TYLOXAPOL® families of compounds.

In certain embodiments the emulsions further comprise one or morecationic halogen containing compounds, including but not limited to,cetylpyridinium chloride. In still further embodiments, the compositionsfurther comprise one or more compounds that increase the interaction(“interaction enhancers”) of the composition with microorganisms (e.g.,chelating agents like ethylenediaminetetraacetic acid, orethylenebis(oxyethylenenitrilo)tetraacetic acid in a buffer).

In some embodiments, the nanoemulsion further comprises an emulsifyingagent to aid in the formation of the emulsion. Emulsifying agentsinclude compounds that aggregate at the oil/water interface to form akind of continuous membrane that prevents direct contact between twoadjacent droplets. Certain embodiments of the present invention featureoil-in-water emulsion compositions that may readily be diluted withwater to a desired concentration without impairing their anti-pathogenicproperties.

In addition to discrete oil droplets dispersed in an aqueous phase,certain oil-in-water emulsions can also contain other lipid structures,such as small lipid vesicles (e.g., lipid spheres that often consist ofseveral substantially concentric lipid bilayers separated from eachother by layers of aqueous phase), micelles (e.g., amphiphilic moleculesin small clusters of 50-200 molecules arranged so that the polar headgroups face outward toward the aqueous phase and the apolar tails aresequestered inward away from the aqueous phase), or lamellar phases(lipid dispersions in which each particle consists of parallelamphiphilic bilayers separated by thin films of water).

These lipid structures are formed as a result of hydrophobic forces thatdrive apolar residues (e.g., long hydrocarbon chains) away from water.The above lipid preparations can generally be described as surfactantlipid preparations (SLPs). SLPs are minimally toxic to mucous membranesand are believed to be metabolized within the small intestine (see e.g.,Hamouda et al., (1998) J. Infect. Disease 180: 1939).

In certain embodiments the emulsion comprises a discontinuous oil phasedistributed in an aqueous phase, a first component comprising an alcoholand/or glycerol, and a second component comprising a surfactant or ahalogen-containing compound. The aqueous phase can comprise any type ofaqueous phase including, but not limited to, water (e.g., dionizedwater, distilled water, tap water) and solutions (e.g., phosphatebuffered saline solution or other buffer systems). The oil phase cancomprise any type of oil including, but not limited to, plant oils(e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseedoil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil,safflower oil, and sunflower oil), animal oils (e.g., fish oil), flavoroil, water insoluble vitamins, mineral oil, and motor oil. In certainembodiments, the oil phase comprises 30-90 vol % of the oil-in-wateremulsion (i.e., constitutes 30-90% of the total volume of the finalemulsion), more preferably 50-80%. The formulations need no be limitedto particular surfactants, however in certain embodiments, thesurfactant is a polysorbate surfactant (e.g., TWEEN 20®, TWEEN 40®,TWEEN 60®, and TWEEN 80®), a pheoxypolyethoxyethanol (e.g., TRITON®X-100, X-301, X-165, X-102, and X-200, and TYLOXAPOL®), or sodiumdodecyl sulfate, and the like.

In certain embodiments a halogen-containing component is present. thenature of the halogen-containing compound, in some preferred embodimentsthe halogen-containing compound comprises a chloride salt (e.g., NaCl,KCl, etc.), a cetylpyridinium halide, a cetyltrimethylammonium halide, acetyldimethylethylammonium halide, a cetyldimethylbenzylammonium halide,a cetyltributylphosphonium halide, dodecyltrimethylammonium halides,tetradecyltrimethylammonium halides, cetylpyridinium chloride,cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride,cetylpyridinium bromide, cetyltrimethylammonium bromide,cetyldimethylethylammonium bromide, cetyltributylphosphonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,and the like

In certain embodiments the emulsion comprises a quaternary ammoniumcompound. Quaternary ammonium compounds include, but are not limited to,N-alkyldimethyl benzyl ammonium saccharinate,1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol; 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride;2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride; alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride; alkyl bis(2-hydroxyethyl)benzyl ammonium chloride; alkyldemethyl benzyl ammonium chloride; alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzylammonium chloride; alkyl dimethyl benzyl ammonium chloride (100% C14);alkyl dimethyl benzyl ammonium chloride (100% C16); alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12); alkyl dimethyl benzylammonium chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12); alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14);alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14); alkyl dimethyl benzylammonium chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18);alkyl dimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;myristalkonium chloride (and) Quat RNIUM 14;N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride; trimethoxysily propyldimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammoniumchloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyldimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethylbenzylammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

Nanoemulsion formulations and methods of making such are well known tothose of skill in the art and described for example in U.S. Pat. Nos.7,476,393, 7,468,402, 7,314,624, 6,998,426, 6,902,737, 6,689,371,6,541,018, 6,464,990, 6,461,625, 6,419,946, 6,413,527, 6,375,960,6,335,022, 6,274,150, 6,120,778, 6,039,936, 5,925,341, 5,753,241,5,698,219, and 5,152,923 and in Fanun et al. (2009) Microemulsions:Properties and Applications (Surfactant Science), CRC Press, Boca RatanFl.

In certain embodiments, one or more active agents described herein canbe provided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water, alcohol, hydrogen peroxide, or otherdiluent.

Extended Release (Sustained Release) Formulations.

In certain embodiments “extended release” formulations of the activeagent(s) described herein are contemplated. In various embodiments suchextended release formulations are designed to avoid the high peak plasmalevels of intravenous and conventional immediate release oral dosageforms.

Illustrative sustained-release formulations include, for example,semipermeable matrices of solid polymers containing the therapeuticagent. Various uses of sustained-release materials have been establishedand are well known by those skilled in the art. Sustained-releasecapsules may, depending on their chemical nature, release the compoundsfor a few weeks up to over 100 days. Depending on the chemical natureand the biological stability of the therapeutic reagent, additionalstrategies for stabilization can be employed.

In certain embodiments such “extended release” formulations utilize themucosa and can independently control tablet disintegration (or erosion)and/or drug dissolution and release from the tablet over time to providea safer delivery profile. In certain embodiments the oral formulationsof active agent(s) described herein (e.g., ASBIs such as galangin,rutin, and analogues, derivatives, or prodrugs thereof, or tautomer(s)or stereoisomer(s) thereof, or pharmaceutically acceptable salts orsolvates of said ASBI(s), said stereoisomer(s), or said tautomer(s), oranalogues, derivatives, or prodrugs thereof) provide individual,repetitive doses that include a defined amount of the active agent thatis delivered over a defined amount of time.

One illustrative sustained release formulation is a substantiallyhomogeneous composition that comprises about 0.01% to about 99% w/w, orabout 0.1% to about 95%, or about 0.1%, or about 1%, or about 2%, orabout 5%, or about 10%, or about 15%, or about 20% to about 80%, or toabout 90%, or to about 95%, or to about 97%, or to about 98%, or toabout 99%1 of the active ingredient(s) (e.g., ASBIs such as galangin,rutin, and analogues, derivatives, or prodrugs thereof, or tautomer(s)or stereoisomer(s) thereof, or pharmaceutically acceptable salts orsolvates of said ASBI(s), said stereoisomer(s), or said tautomer(s), oranalogues, derivatives, or prodrugs thereof) and one or moremucoadhesives (also referred to herein as “bioadhesives”) that providefor adherence to the targeted mucosa of the subject (patient) and thatmay further comprise one or more of the following: one or more bindersthat provide binding of the excipients in a single tablet; one or morehydrogel forming excipients; one or more bulking agents; one or morelubricants; one or more glidants; one or more solubilizers; one or moresurfactants; one or more flavors; one or more disintegrants; one or morebuffering excipients; one or more coatings; one or more controlledrelease modifiers; and one or more other excipients and factors thatmodify and control the drug's dissolution or disintegration time andkinetics or protect the active drug from degradation.

In various embodiments a sustained release pharmaceutical dosage formfor oral transmucosal delivery can be solid or non-solid. In onepreferred embodiment, the dosage from is a solid that turns into ahydrogel following contact with saliva.

Suitable excipients include, but are not limited to substances added tothe formulations that are required to produce a commercial product andcan include, but are not limited to: bulking agents, binders,surfactants, bioadhesives, lubricants, disintegrants, stabilizers,solubilizers, glidants, and additives or factors that affect dissolutionor disintegration time. Suitable excipients are not limited to thoseabove, and other suitable nontoxic pharmaceutically acceptable carriersfor use in oral formulations can be found in Remington's PharmaceuticalSciences, 17th Edition, 1985.

In certain embodiments extended release formulations of the activeagent(s) described herein for oral transmucosal drug delivery include atleast one bioadhesive (mucoadhesive) agent or a mixture of severalbioadhesives to promote adhesion to the oral mucosa during drugdelivery. In addition the bioadhesive agents may also be effective incontrolling the dosage form erosion time and/or, the drug dissolutionkinetics over time when the dosage form is wetted. Such mucoadhesivedrug delivery systems are very beneficial, since they can prolong theresidence time of the drug at the site of absorption and increase drugbioavailability. The mucoadhesive polymers forming hydrogels aretypically hydrophilic and swellable, containing numerous hydrogenbond-forming groups, like hydroxyl, carboxyl or amine, which favoradhesion. When used in a dry form, they attract water from the mucosalsurface and swell, leading to polymer/mucus interaction through hydrogenbonding, electrostatic, hydrophobic or van der Waals interaction.

Illustrative suitable mucoadhesive or bioadhesive materials, include,but are not limited to natural, synthetic or biological polymers,lipids, phospholipids, and the like. Examples of natural and/orsynthetic polymers include cellulosic derivatives (such asmethylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose,hydroxyethylmethyl cellulose, etc), natural gums (such as guar gum,xanthan gum, locust bean gum, karaya gum, veegum etc.), polyacrylates(such as CARBOPOL®, polycarbophil, etc), alginates, thiol-containingpolymers, POLYOX®yethylenes, polyethylene glycols (PEG) of all molecularweights (preferably between 1000 and 40,000 Da, of any chemistry, linearor branched), dextrans of all molecular weights (preferably between 1000and 40,000 Da of any source), block copolymers, such as those preparedby combinations of lactic and glycolic acid (PLA, PGA, PLGA of variousviscosities, molecular weights and lactic-to-glycolic acid ratios)polyethylene glycol-polypropylene glycol block copolymers of any numberand combination of repeating units (such as PLURONICS®, TEKTRONIX® orGENAPOL® block copolymers), combination of the above copolymers eitherphysically or chemically linked units (for example PEG-PLA or PEG-PLGAcopolymers) mixtures. Preferably the bioadhesive excipient is selectedfrom the group of polyethylene glycols, POLYOX®yethylenes, polyacrylicacid polymers, such as CARBOPOL® (such as CARBOPOL® 71G, 934P, 971P,974P, and the like) and polycarbophils (such as NOVEON® AA-1, NOVEON®CA-1, NOVEON® CA-2, and the like), cellulose and its derivatives andmost preferably it is polyethylene glycol, carbopol, and/or a cellulosicderivative or a combination thereof.

In certain embodiments the mucoadhesive/bioadhesive excipient istypically present at 1-50% w/w, preferably 1-40% w/w or most preferablybetween 5-30% w/w. A particular formulation may contain one or moredifferent bioadhesives in any combination.

In certain embodiments the formulations for oral transmucosal drugdelivery also include a binder or mixture of two or more binders whichfacilitate binding of the excipients into a single dosage form.Exemplary binders are selected from the group consisting of cellulosicderivatives (such as methylcellulose, carboxymethyl cellulose,hydroxyethyl cellulose, hydroxyethylmethyl cellulose, etc.),polyacrylates (such as CARBOPOL®, polycarbophil, etc.), POVIDONE® (allgrades), POLYOX®® of any molecular weight or grade, irradiated or not,starch, polyvinylpyrrolidone (PVP), AVICEL®, and the like. In certainembodiments the binder is typically present at 0.5-60% w/w, preferably1-30% w/w and most preferably 1.5-15% w/w.

In certain embodiments the formulations also include at least onehydrogel-forming excipient. Exemplary hydrogel forming excipients areselected from the group consisting of polyethylene glycols and otherpolymers having an ethylene glycol backbone, whether homopolymers orcross linked heteropolymers, block copolymers using ethylene glycolunits, such as POLYOX®yethylene homopolymers (such as POLYOX®®N10/MW=100,000 POLYOX®-80/MW=200,000; POLYOX® 1105/MW=900,000;POLYOX®-301/MW=4,000,000; POLYOX®-303/MW=7,000,000, POLYOX® WSR-N-60K,all of which are tradenames of Union Carbide),hydroxypropylmethylcellylose (HPMC) of all molecular weights and grades(such as METOLOSE® 90SH50000, METOLOSE® 90SH30000, all of which aretradenames of Shin-Etsu Chemical company), Poloxamers (such as LUTROL®F-68, LUTROL® F-127, F-105 etc., all tradenames of BASF Chemicals),GENAPOL®, polyethylene glycols (PEG, such as PEG-1500, PEG-3500,PEG-4000, PEG-6000, PEG-8000, PEG-12000, PEG-20,000, etc.), natural gums(xanthan gum, locust bean gum, etc.) and cellulose derivatives (HC, HMC,HMPC, HPC, CP, CMC), polyacrylic acid-based polymers either as free orcross-linked and combinations thereof, biodegradable polymers such aspoly lactic acids, polyglycolic acids and any combination thereof,whether a physical blend or cross-linked. In certain embodiments, thehydrogel components may be cross-linked. The hydrogel formingexcipient(s) are typically present at 0.1-70% w/w, preferably 1-50% w/wor most preferably 1-30% w/w.

In certain embodiments the formulations may also include at least onecontrolled release modifier which is a substance that upon hydration ofthe dosage form will preferentially adhere to the drug molecules andthus reduce the rate of its diffusion from the oral dosage form. Suchexcipients may also reduce the rate of water uptake by the formulationand thus enable a more prolonged drug dissolution and release from thetablet. In general the selected excipient(s) are lipophilic and capableof naturally complexing to the hydrophobic or lipophilic drugs. Thedegree of association of the release modifier and the drug can be variedby altering the modifier-to-drug ratio in the formulation. In addition,such interaction may be appropriately enhanced by the appropriatecombination of the release modifier with the active drug in themanufacturing process. Alternatively, the controlled release modifiermay be a charged polymer either synthetic or biopolymer bearing a netcharge, either positive or negative, and which is capable of binding tothe active via electrostatic interactions thus modifying both itsdiffusion through the tablet and/or the kinetics of its permeationthrough the mucosal surface. Similarly to the other compounds mentionedabove, such interaction is reversible and does not involve permanentchemical bonds with the active. In certain embodiments the controlledrelease modifier may typically be present at 0-80% w/w, preferably 1-20%w/w, most preferably 1-10% w/w.

In various embodiments the extended release formulations may alsoinclude other conventional components required for the development oforal dosage forms, which are known to those skilled in the art. Thesecomponents may include one or more bulking agents (such as lactose USP,Starch 1500, mannitol, sorbitol, malitol or other non-reducing sugars;microcrystalline cellulose (e.g., AVICEL®), dibasic calcium phosphatedehydrate, sucrose, and mixtures thereof), at least one solubilizingagent(s) (such as cyclodextrins, pH adjusters, salts and buffers,surfactants, fatty acids, phospholipids, metals of fatty acids etc.),metal salts and buffers organic (such as acetate, citrate, tartrate,etc.) or inorganic (phosphate, carbonate, bicarbonate, borate, sulfate,sulfite, bisulfite, metabisulfite, chloride, etc.), salts of metals suchas sodium, potassium, calcium, magnesium, etc.), at least one lubricant(such as stearic acid and divalent cations of, such as magnesiumstearate, calcium stearate, etc., talc, glycerol monostearate and thelike), one or more glidants (such as colloidal silicon dioxide,precipitated silicon dioxide, fumed silica (CAB-O-SIL® M-5P, trademarkof Cabot Corporation), stearowet and sterotex, silicas (such as SILOID®and SILOX® silicas—trademarks of Grace Davison Products,Aerosil—trademark of Degussa Pharma), higher fatty acids, the metalsalts thereof, hydrogenated vegetable oils and the like), flavors orsweeteners and colorants (such as aspartame, mannitol, lactose, sucrose,other artificial sweeteners; ferric oxides and FD&C lakes), additives tohelp stabilize the drug substance from chemical of physical degradation(such as anti-oxidants, anti-hydrolytic agents, aggregation-blockersetc. Anti-oxidants may include BHT, BHA, vitamins, citric acid, EDTA,sodium bisulfate, sodium metabisulfate, thiourea, methionine,surfactants, amino-acids, such as arginine, glycine, histidine,methionine salts, pH adjusters, chelating agents and buffers in the dryor solution form), one or more excipients that may affect tabletdisintegration kinetics and drug release from the tablet, and thuspharmacokinetics (disintegrants such as those known to those skilled inthe art and may be selected from a group consisting of starch,carboxy-methylcellulose type or crosslinked polyvinyl pyrrolidone (suchas cross-povidone, PVP-XL), alginates, cellulose-based disintegrants(such as purified cellulose, methylcellulose, crosslinked sodium carboxymethylcellulose (Ac-Di-Sol) and carboxy methyl cellulose), lowsubstituted hydroxypropyl ethers of cellulose, microcrystallinecellulose (such as AVICEL®), ion exchange resins (such as AMBRELITE® IPR88), gums (such as agar, locust bean, karaya, pectin and tragacanth),guar gums, gum karaya, chitin and chitosan, smecta, gellan gum,isapghula husk, polacrillin potassium (Tulsion ³³⁹) gas-evolvingdisintegrants (such as citric acid and tartaric acid along with thesodium bicarbonate, sodium carbonate, potassium bicarbonate or calciumcarbonate), sodium starch glycolate (such as EXPLOTAB® and PRIMOGEL®),starch DC and the likes, at least one biodegradable polymer of any typeuseful for extended drug release. Exemplary polymer compositionsinclude, but are not limited to, polyanhydrides and co-polymers oflactic acid and glycolic acid, poly(dl-lactide-co-glycolide) (PLGA),poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polyorthoesters,proteins, and polysaccharides.

In certain embodiments, the active agent(s) can be chemically modifiedto significantly modify the pharmacokinetics in plasma. This may beaccomplished for example by conjugation with poly(ethylene glycol)(PEG), including site-specific PEGylation. PEGylation, which may improvedrug performance by optimizing pharmacokinetics, decreasingimmunogenicity and dosing frequency.

Methods of making a formulation of the active agent(s) described herein(e.g., ASBIs such as galangin, rutin, and analogues, derivatives, orprodrugs thereof) for GI or oral transmucosal delivery are alsoprovided. One method includes the steps of powder grinding, dry powdermixing and tableting via direct compression. Alternatively, a wetgranulation process may be used. Such a method (such as high sheargranulation process) involves mixing the active ingredient and possiblysome excipients in a mixer. The binder may be one of the excipientsadded in the dry mix state or dissolved in the fluid used forgranulating. The granulating solution or suspension is added to the drypowders in the mixer and mixed until the desired characteristics areachieved. This usually produces a granule that will be of suitablecharacteristics for producing dosage forms with adequate dissolutiontime, content uniformity, and other physical characteristics. After thewet granulation step, the product is most often dried and/or then milledafter drying to get a major percentage of the product within a desiredsize range. Sometimes, the product is dried after being wet sized usinga device such as an oscillating granulator, or a mill. The drygranulation may then processed to get an acceptable size range by firstscreening with a sieving device, and then milling the oversizedparticles.

Additionally, the formulation may be manufactured by alternativegranulation processes, all known to those skilled in the art, such asspray fluid bed granulation, extrusion and spheronization or fluid bedrotor granulation.

Additionally, the tablet dosage form of the invention may be prepared bycoating the primary tablet manufactured as described above with suitablecoatings known in the art. Such coatings are meant to protect the activecores against damage (abrasion, breakage, dust formation) againstinfluences to which the cores are exposed during transport and storage(atmospheric humidity, temperature fluctuations), and naturally thesefilm coatings can also be colored. The sealing effect of film coatsagainst water vapor is expressed by the water vapor permeability.Coating may be performed by one of the available processes such asWurster coating, dry coating, film coating, fluid bed coating, pancoating, etc. Typical coating materials include polyvinyl pyrrolidone(PVP), polyvinyl pyrrolidone vinyl acetate copolymer (PVPVA), polyvinylalcohol (PVA), polyvinyl alcohol/polyethylene glycol copolymer(PVA/PEG), cellulose acetate phthalate, ethyl cellulose, gellan gum,maltodextrin, methacrylates, methyl cellulose, hydroxyl propyl methylcellulose (HPMC of all grades and molecular weights), carrageenan,shellac and the like.

In certain embodiments the tablet core comprising the active agent(s)described herein can be coated with a bioadhesive and/or pH resistantmaterial to enable material, such as those defined above, to improvebioadhesion of the tablet in the sublingual cavity.

In certain embodiments, the active agent(s) described herein (e.g.,ASBIs such as galangin, rutin, and analogues, derivatives, or prodrugsthereof) are formulated as inclusion complexes. While not limited tocyclodextrin inclusion complexes, it is noted that cyclodextrin is theagent most frequently used to form pharmaceutical inclusion complexes.Cyclodextrins (CD) are cyclic oligomers of glucose, that typicallycontain 6, 7, or 8 glucose monomers joined by α-1,4 linkages. Theseoligomers are commonly called α-CD, β-CD, and γ-CD, respectively. Higheroligomers containing up to 12 glucose monomers are known, andcontemplated to in the formulations described herein. Functionalizedcyclodextrin inclusion complexes are also contemplated. Illustrative,but non-limiting functionalized cyclodextrins include, but are notlimited to sulfonates, sulfonates and sulfinates, or disulfonates ofhydroxybutenyl cyclodextrin; sulfonates, sulfonates and sulfinates, ordisulfonates of mixed ethers of cyclodextrins where at least one of theether substituents is hydroxybutenyl cyclodextrin. Illustrativecyclodextrins include a polysaccharide ether which comprises at leastone 2-hydroxybutenyl substituent, wherein the at least onehydroxybutenyl substituent is sulfonated and sulfinated, ordisulfonated, and an alkylpolyglycoside ether which comprises at leastone 2-hydroxybutenyl substituent, wherein the at least onehydroxybutenyl substituent is sulfonated and sulfinated, ordisulfonated. In various embodiments inclusion complexes formed betweensulfonated hydroxybutenyl cyclodextrins and one or more of the activeagent(s) described herein are contemplated. Methods of preparingcyclodextrins, and cyclodextrin inclusion complexes are found forexample in U.S. Patent Publication No: 2004/0054164 and the referencescited therein and in U.S. Patent Publication No: 2011/0218173 and thereferences cited therein.

Pharmacokinetics (PK) and Formulation Attributes

One advantage of the extended (controlled) release oral (GI ortransmucosal) formulations described herein is that they can maintainthe plasma drug concentration within a targeted therapeutic window for alonger duration than with immediate-release formulations, whether soliddosage forms or liquid-based dosage forms. The high peak plasma levelstypically observed for such conventional immediate release formulationswill be blunted by the prolonged release of the drug over 1 to 12 hoursor longer. In addition, a rapid decline in plasma levels will be avoidedsince the drug will continually be crossing from the oral cavity intothe bloodstream during the length of time of dissolution of the tablet,thus providing plasma pharmacokinetics with a more stable plateau. Inaddition, the dosage forms described herein may improve treatment safetyby minimizing the potentially deleterious side effects due to thereduction of the peaks and troughs in the plasma drug pharmacokinetics,which compromise treatment safety.

In various embodiments the oral transmucosal formulations of the activeagent(s) described herein designed to avoid the high peak plasma levelsof intravenous and conventional immediate release oral dosage forms byutilizing the mucosa and by independently controlling both tabletdisintegration (or erosion) and drug dissolution and release from thetablet over time to provide a safer delivery profile. The oralformulations described herein provide individual, repetitive doses thatinclude a defined amount of the active agent.

An advantage of the bioadhesive oral transmucosal formulations describedherein is that they exhibit highly consistent bioavailability and canmaintain the plasma drug concentration within a targeted therapeuticwindow with significantly lower variability for a longer duration thancurrently available dosage forms, whether solid dosage forms or IVdosage forms. In addition, a rapid decline in plasma levels is avoidedsince the drug is continually crossing from the oral cavity or GI tractinto the bloodstream during the length of time of dissolution of thetablet or longer, thus providing plasma pharmacokinetics with anextended plateau phase as compared to the conventional immediate releaseoral dosage forms. Further, the dosage forms described herein canimprove treatment safety by minimizing the potentially deleterious sideeffects due to the relative reduction of the peaks and troughs in theplasma drug pharmacokinetics, which compromise treatment safety and istypical of currently available dosage forms.

In various embodiments bioadhesive formulations described herein can bedesigned to manipulate and control the pharmacokinetic profile of theactive agent(s) described herein. As such, the formulations can beadjusted to achieve ‘slow’ disintegration times (and erosion kineticprofiles) and slow drug release and thus enable very prolongedpharmacokinetic profiles that provide sustained drug action. Althoughsuch formulations may be designed to still provide a fast onset, theyare mostly intended to enable the sustained drug PK and effect whilemaintaining the other performance attributes of the tablet such asbioadhesion, reproducibility of action, blunted C_(max), etc.

The performance and attributes of the bioadhesive transmucosalformulations of this invention are independent of the manufacturingprocess. A number of conventional, well-established and known in the artprocesses can be used to manufacture the formulations of the presentinvention (such as wet and dry granulation, direct compression, etc.)without impacting the dosage form physicochemical properties or in vivoperformance.

An illustrative mathematical ratio that demonstrates the prolongedplateau phase of the measured blood plasma levels of the active agent(s)described herein, following administration of the dosage forms of theinvention is the term “Optimal Therapeutic Targeting Ratio” or “OTTR”,which represents the average time that the drug is present attherapeutic levels, defined as time within which the drug plasmaconcentration is maintained above 50% of C_(max) normalized by thedrug's elimination half-life multiplied by the ratio of the C_(max)obtained in the dosage form of interest over the normalized C. followingIV administration of equivalent doses. In certain embodiments the OTTRcan be calculated by the formula:OTTR=(C ^(IV) _(max) /C _(max))×(Dose/Dose^(IV))(Time above 50% of C_(max))/(Terminal^(IV) elimination half-life of the drug).In certain embodiments the OTTR is greater than about 15, or greaterthan about 20, or greater than about 25, or greater than about 30, orgreater than about 40, or greater than about 50.Administration

In certain embodiments one or more active agents described herein (e.g.,ASBIs such as galangin, rutin, and analogues, derivatives, or prodrugsthereof) are administered to a mammal in need thereof, e.g., to a mammalat risk for or suffering from a pathology characterized by abnormalprocessing of amyloid precursor proteins, a mammal at risk forprogression of MCI to Alzheimer's disease, and so forth. In certainembodiments the active agent(s) are administered to prevent or delay theonset of a pre-Alzheimer's cognitive dysfunction, and/or to ameliorateone or more symptoms of a pre-Alzheimer's cognitive dysfunction, and/orto prevent or delay the progression of a pre-Alzheimer's condition orcognitive dysfunction to Alzheimer's disease, and/or to promote theprocessing of amyloid precursor protein (APP) by a non-amyloidogenicpathway. In certain embodiments one or more active agent(s) areadministered for the treatment of early stage, mid stage, or late-stageAlzheimer's disease, e.g., to reduce the severity of the disease, and/orto ameliorate one or more symptoms of the disease, and/or to slow theprogression of the disease.

In various embodiments the active agent(s) described herein (e.g., ASBIssuch as galangin, rutin, and analogues, derivatives, or prodrugsthereof) can be administered by any of a number of routes. Thus, forexample they can be administered orally, parenterally, (intravenously(IV), intramuscularly (IM), depo-IM, subcutaneously (SQ), and depo-SQ),sublingually, intranasally (inhalation), intrathecally, transdermally(e.g., via transdermal patch), topically, ionophoretically or rectally.Typically the dosage form is selected to facilitate delivery to thebrain (e.g., passage through the blood brain barrier). In this contextit is noted that the compounds described herein are readily delivered tothe brain. Dosage forms known to those of skill in the art are suitablefor delivery of the compound.

The active agent(s) are administered in an amount/dosage regimensufficient to exert a prophylactically and/or therapeutically usefuleffect in the absence of undesirable side effects on the subjecttreated. The specific amount/dosage regimen will vary depending on theweight, gender, age and health of the individual; the formulation, thebiochemical nature, bioactivity, bioavailability and the side effects ofthe particular compound.

In certain embodiments the therapeutically or prophylactically effectiveamount may be determined empirically by testing the agent(s) in known invitro and in vivo model systems for the treated disorder. Atherapeutically or prophylactically effective dose can be determined byfirst administering a low dose, and then incrementally increasing untila dose is reached that achieves the desired effect with minimal or noundesired side effects.

In certain embodiments, when administered orally, an administered amountof the agent(s) described herein effective to prevent or delay the onsetof a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one ormore symptoms of a pre-Alzheimer's cognitive dysfunction, and/or toprevent or delay the progression of a pre-Alzheimer's condition orcognitive dysfunction to Alzheimer's disease, and/or to promote theprocessing of amyloid precursor protein (APP) by a non-amyloidogenicpathway, and/or to treat or prevent AD ranges from about 0.1 mg/day toabout 500 mg/day or about 1,000 mg/day, or from about 0.1 mg/day toabout 200 mg/day, for example, from about 1 mg/day to about 100 mg/day,for example, from about 5 mg/day to about 50 mg/day. In someembodiments, the subject is administered the compound at a dose of about0.05 to about 0.50 mg/kg, for example, about 0.05 mg/kg, 0.10 mg/kg,0.20 mg/kg, 0.33 mg/kg, 0.50 mg/kg. It is understood that while apatient may be started at one dose, that dose may be varied (increasedor decreased, as appropriate) over time as the patient's conditionchanges. Depending on outcome evaluations, higher doses may be used. Forexample, in certain embodiments, up to as much as 1000 mg/day can beadministered, e.g., 5 mg/day, 10 mg/day, 25 mg/day, 50 mg/day, 100mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, 600 mg/day, 700mg/day, 800 mg/day, 900 mg/day or 1000 mg/day.

In various embodiments, active agent(s) described herein can beadministered parenterally, for example, by IV, IM, depo-IM, SC, ordepo-SC. When administered parenterally, a therapeutically effectiveamount of about 0.5 to about 100 mg/day, preferably from about 5 toabout 50 mg daily should be delivered. When a depot formulation is usedfor injection once a month or once every two weeks, the dose should beabout 0.5 mg/day to about 50 mg/day, or a monthly dose of from about 15mg to about 1,500 mg. In part because of the forgetfulness of thepatients with Alzheimer's disease, it is preferred that the parenteraldosage form be a depo formulation.

In various embodiments, the active agent(s) described herein can beadministered sublingually. When given sublingually, the compounds and/oranalogs thereof can be given one to four times daily in the amountsdescribed above for IM administration.

In various embodiments, the active agent(s) described herein can beadministered intranasally. When given by this route, the appropriatedosage forms are a nasal spray or dry powder, as is known to thoseskilled in the art. The dosage of compound and/or analog thereof forintranasal administration is the amount described above for IMadministration.

In various embodiments, the active agent(s) described herein can beadministered intrathecally. When given by this route the appropriatedosage form can be a parenteral dosage form as is known to those skilledin the art. The dosage of compound and/or analog thereof for intrathecaladministration is the amount described above for IM administration.

In certain embodiments, the active agent(s) described herein can beadministered topically. When given by this route, the appropriate dosageform is a cream, ointment, or patch. When administered topically, thedosage is from about 1.0 mg/day to about 200 mg/day. Because the amountthat can be delivered by a patch is limited, two or more patches may beused. The number and size of the patch is not important, what isimportant is that a therapeutically effective amount of compound bedelivered as is known to those skilled in the art. The compound can beadministered rectally by suppository as is known to those skilled in theart. When administered by suppository, the therapeutically effectiveamount is from about 1.0 mg to about 500 mg.

In various embodiments, the active agent(s) described herein can beadministered by implants as is known to those skilled in the art. Whenadministering the compound by implant, the therapeutically effectiveamount is the amount described above for depot administration.

In various embodiments the dosage forms can be administered to thesubject 1, 2, 3, or 4 times daily. It is preferred that the compound beadministered either three or fewer times, more preferably once or twicedaily. It is preferred that the agent(s) be administered in oral dosageform.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular conditionbeing treated, the severity of the condition being treated, the age,weight, general physical condition of the particular patient, and othermedication the individual may be taking as is well known toadministering physicians who are skilled in this art.

While the compositions and methods are described herein with respect touse in humans, they are also suitable for animal, e.g., veterinary use.Thus certain preferred organisms include, but are not limited to humans,non-human primates, canines, equines, felines, porcines, ungulates,largomorphs, and the like.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

Combination Therapies

In certain embodiments, the active agent(s) described herein (e.g.,ASBIs such as galangin, rutin, and analogues, derivatives, or prodrugsthereof) can be used in combination with other therapeutic agents orapproaches used to treat or prevent diseases characterized by amyloiddeposits in the brain, including MCI and/or AD. Such agents orapproaches include: acetylcholinesterase inhibitors (including withoutlimitation, e.g., (−)-phenserine enantiomer, tacrine, ipidacrine,galantamine, donepezil, icopezil, zanapezil, rivastigmine, huperzine A,phenserine, physostigmine, neostigmine, pyridostigmine, ambenonium,demarcarium, edrophonium, ladostigil and ungeremine); NMDA receptorantagonist (including without limitations e.g., Memantine); muscarinicreceptor agonists (including without limitation, e.g., Talsaclidine,AF-102B, AF-267B (NGX-267)); nicotinic receptor agonists (includingwithout limitation, e.g., Ispronicline (AZD-3480)); beta-secretaseinhibitors (including without limitations e.g., thiazolidinediones,including rosiglitazone and pioglitazone); gamma-secretase inhibitors(including without limitation, e.g., semagacestat (LY-450139), MK-0752,E-2012, BMS-708163, PF-3084014, begacestat (GSI-953), and NIC5-15);inhibitors of Aβ aggregation (including without limitation, e.g.,Clioquinol (PBT1), PBT2, tramiprosate (homotaurine), Scyllo-inositol(a.k.a., scyllo-cyclohexanehexol, AZD-103 and ELND-005), passiveimmunotherapy with Aβ fragments (including without limitations e.g.,Bapineuzemab) and Epigallocatechin-3-gallate (EGCg)); anti-inflammatoryagents such as cyclooxygenase II inhibitors; anti-oxidants such asVitamin E and ginkolides; immunological approaches, such as, forexample, immunization with Aβ peptide or administration of anti-Aβpeptide antibodies; statins; and direct or indirect neurotrophic agentssuch as Cerebrolysin™, AIT-082 (Emilieu (2000) Arch. Neurol. 57:454),Netrin (Luorenco (2009) Cell Death Differ 16: 655-663), Netrin mimetics,NGF, NGF mimetics, BDNF and other neurotrophic agents of the future,agents that promote neurogenesis e.g. stem cell therapy. Furtherpharmacologic agents useful in combination with tropisetron, disulfiram,honokiol and/or nimetazepam to treat or prevent diseases characterizedby amyloid deposits in the brain, including MCI and/or AD, aredescribed, e.g., in Mangialasche, et al., Lancet Neurol (2010)9:702-716.

In various embodiments, combination therapy with one or more of theactive agents described herein expressly excludes administration ofthese agents in conjunction with an acetylcholinesterase inhibitor.

Use of ASBIs in Age Related Macular Degeneration and Glaucoma.

While in various embodiments, the use of ASBIs are contemplated for thepreventing or delaying the onset of a pre-Alzheimer's condition and/orcognitive dysfunction, and/or ameliorating one or more symptoms of apre-Alzheimer's condition and/or cognitive dysfunction, or preventing ordelaying the progression of a pre-Alzheimer's condition or cognitivedysfunction to Alzheimer's disease, and/or for the treatment ofAlzheimer's disease, other uses of ASBIs are also contemplated. Inparticular, in certain embodiments, the use of ASBIs is contemplated forthe treatment and/or prophylaxis of age-related macular degenerationand/or glaucoma.

Without being bound to a particular theory, it is believed that abnormalextracellular deposition of proteins may contribute to age-relatedmacular degeneration (AMD) pathogenesis and progression, which is alsothe case in Alzheimer's disease and atherosclerosis. In both conditions,the protein deposits contain many shared constituents such as apoE,complement, and Aβ peptides. For instance, in human AMD, Aβ peptidedeposition is associated with drusen, where it accumulates andcolocalizes with activated complement components (Anderson et al. (2004)Exp. Eye. Res., 78:243-256; Dentchev et al. (2003) Mol. Vis., 9:184-190; Johnson et al. (2002) Proc Natl Acad Sci USA 99: 11830-11835).Luibl et al. (2006) J. Clin. Invest., 116: 378-385, showed the presenceof potentially toxic amyloid oligomers in drusen, sub-RPE basaldeposits, and RPE of human donor eyes using an antibody thatspecifically recognizes the oligomeric form of Aβ. These Aβ oligomerswere not detected in control age-matched donor eyes without drusen. Isaset al. (2010) Invest. Ophthalmol Vis. Sci., 51: 1304-1310, also detectedsoluble as well as mature Aβ fibrils in drusen. Collectively, thesefindings implicate Aβ in the pathogenesis of AMD. In addition, Aβpeptide has been detected in sub-RPE basal deposits and neovascularlesions in a murine model of AMD (Ding et al. (2008) Vision Res., 48:339-345; Malek et al. (2005) Proc Natl Acad Sci USA, 102: 11900-11905).In this model, aged human APOE4-targeted replacement mice (APOE4 mice)fed a high-fat, cholesterol-enriched (HFC) diet (APOE4-HFC mice) exhibitmorphologic hallmarks observed in both dry and wet AMD. These hallmarksinclude thick diffuse sub-RPE deposits, lipid- and protein-containingfocal drusen-like deposits, thickening of Bruch's membrane, patchyregions of RPE atrophy opposed to areas of photoreceptor degeneration,and CNV (Malek et al. (2005) Proc Natl Acad Sci USA, 102: 11900-11905).It is believed that, in the APOE4-HFC mouse model of AMD, Aβaccumulation provokes damage at the level of the RPE/choroid and haspreviously been shown that systemic administration of anti-Aβ40-specificantibodies can partially attenuate the decline in visual functionexhibited in this model (Ding et al. (2008) Vision Res., 48: 339-345).It has also been demonstrated that anti-Aβ immunotherapy simultaneouslytargeting both Aβ40 and Aβ42 blocks histopathologic changes andcompletely protects visual function in APOE4-HFC mice (Ding et al.(2011) Proc. Nat'l. Acad. Sci. U.S.A., 108 (28): E279-E287).

Without being bound by a particular theory, it is believed that APPprocessing to Aβ in the eye occurs by the activities of BACE andγ-secretase in the retina and retinal pigmented epithelial (RPE) celllayers and that sAPPα and Aβ are secreted into the vitreous humor (see,e.g., (Prakasam et al. (2008) J. Alzh. Dis., 20: 1243-1253). Aβ isfurther transported into the aqueous humor where it is readily measured.

In view of these findings, it is believe that ASBIs, e.g., as describedherein, can find use in the treatment or prophylaxis of age-relatedmacular degeneration (AMD) and/or glaucoma. Accordingly, it is believedthat ASBIs can be administered to a subject to slow or prevent theappearance of AMD (and/or glaucoma), and/or to reduce one or moresymptoms of AMD, and/or to slow, stop, or reverse progression of thedisease. In various embodiments one or more ASBIs (e.g., any one or moreof the active agent(s) described herein) are administered to a subject(e.g., a human, a non-human mammal) for these purposes. As describedabove, in various embodiments, the ASBI is administered via a routeselected from the group consisting of oral delivery, isophoreticdelivery, transdermal delivery, parenteral delivery, aerosoladministration, administration via inhalation, intravenousadministration, and rectal administration.

In certain embodiments, the administration is directly to the eye. Thusfor example, in certain embodiments, the agent(s) can be administered tothe eye in the form of eye drops, via intraocular injection, and thelike.

Typically the ASBIs are administered in an effective amount for thetreatment and/or prophylaxis of AMD or glaucoma, where the effectiveamount will vary by the modality of administration. In certainembodiments effective amount is an amount sufficient to mitigating in amammal one or more symptoms associated with age-related maculardegeneration (AMD). In certain embodiments the effective amount is anamount, an amount sufficient to reduce the risk or delaying the onset,and/or reduce the ultimate severity of a AMD disease (or glaucoma)characterized by reduction of Aβ in the vitreous and/or aqueous humorand/or the amyloid deposits on the retina and/or the RPE cell layer.

Assay Systems to Evaluate APP Processing

Without being bound to a particular theory, it is believed that theactive agent(s) described herein (e.g., ASBIs such as galangin, rutin,and analogues, derivatives, or prodrugs thereof) promote processing ofAPP by the nonamyloidogenic pathway and/or reduce or inhibits processingof APP by the amyloidogenic pathway. In the nonamyloidogeic pathway, APPis first cleaved by α-secretase within the Aβ sequence, releasing theAPPsα ectodomain (“sAPPα”). In contrast, the amyloidogenic pathway isinitiated when β-secretase cleaves APP at the amino terminus of the Aβ,thereby releasing the APPsβ ectodomain (“sAPPβ”). APP processing by thenonamyloidogenic and amyloidogenic pathways is known in the art andreviewed, e.g., by Xu (2009) J Alzheimers Dis., 16(2): 211-224, and DeStrooper, et al. (2010 Nat Rev Neurol 6(2): 99-107.

One method to evaluate the efficacy of the active agent(s) is todetermine a reduction or elimination in the level of APP processing bythe amyloidogenic pathway, e.g., a reduction or elimination in the levelof APP processing by β-secretase cleavage in response to theadministration of the agent(s) of interest. Assays for determining theextent of APP cleavage at the β-secretase cleavage site are well knownin the art. Illustrative assays are described, for example, in U.S. Pat.Nos. 5,744,346 and 5,942,400. Kits for determining the presence andlevels in a biological sample of sAPPα and sAPPβ, as well as APPneo andAβ commercially available, e.g., from PerkinElmer.

ASBI Assay.

ASBI activity of any of the compounds described herein can readily beverified using, for example, assays illustrated in the examples providedherein. Basically, in certain embodiments a pair of assays are utilizedto identify compounds that inhibit BACE cleavage of the MBP-C125 APPsubstrate, resulting in the inhibition of the production of C99 but notthe β-site peptide substrate (P5-P5′).

As illustrated in the Examples, in one embodiment, an MBP-C125 APP695 wtfusion protein can be used as one of the substrates. The secondsubstrate can be the commercially available P5-P5′ fluorescencesubstrate. Each of these substrates is incubated with recombinant BACE(R&D (cat#931-AS-050) in, for example, a 96 well plate format. For theMBP-C125 substrate the C-99 product from the BACE cleavage can bemeasured using an AlphaLisa assay as a readout. For the P5-5′ substratethe loss of fluorescence upon BACE cleavage can be used as the readout.An ASBI would inhibit the BACE cleavage of the MBP-C125 substrate whilenot being inhibitory of the fluorescence substrate.

Other Cell Free Assays

Illustrative assays that can be used to demonstrate the inhibitoryactivity of the active agent(s) are described, for example, in WO00/17369, WO 00/03819, and U.S. Pat. Nos. 5,942,400 and 5,744,346. Suchassays can be performed in cell-free incubations or in cellularincubations using cells expressing an alpha-secretase and/orbeta-secretase and an APP substrate having a alpha-secretase andbeta-secretase cleavage sites.

In one illustrative embodiment, the agent(s) of interest are contactedwith an APP substrate containing alpha-secretase and beta-secretasecleavage sites of APP, for example, a complete APP or variant, an APPfragment, or a recombinant or synthetic APP substrate containing theamino acid sequence: KM-DA or NL-DA (APP-SW), is incubated in thepresence of an alpha-secretase and/or beta-secretase enzyme, a fragmentthereof, or a synthetic or recombinant polypeptide variant havingalpha-secretase or beta-secretase activity and effective to cleave thealpha-secretase or beta-secretase cleavage sites of APP, underincubation conditions suitable for the cleavage activity of the enzyme.agent(s) having the desired activity reduce or prevent cleavage of theAPP substrate. Suitable substrates optionally include derivatives thatmay be fusion proteins or peptides that contain the substrate peptideand a modification useful to facilitate the purification or detection ofthe peptide or its alpha-secretase and/or beta-secretase cleavageproducts. Useful modifications include the insertion of a knownantigenic epitope for antibody binding; the linking of a label ordetectable moiety, the linking of a binding substrate, and the like.

Suitable incubation conditions for a cell-free in vitro assay include,for example: approximately 200 nanomolar to 10 micromolar substrate,approximately 10 to 200 picomolar enzyme, and approximately 0.1nanomolar to 10 micromolar of the agent(s), in aqueous solution, at anapproximate pH of 4-7, at approximately 37° C., for a time period ofapproximately 10 minutes to 3 hours. These incubation conditions areexemplary only, and can be varied as required for the particular assaycomponents and/or desired measurement system. Optimization of theincubation conditions for the particular assay components should accountfor the specific alpha-secretase and/or beta-secretase enzyme used andits pH optimum, any additional enzymes and/or markers that might be usedin the assay, and the like. Such optimization is routine and will notrequire undue experimentation.

Another illustrative assay utilizes a fusion peptide having maltosebinding protein (MBP) fused to the C-terminal 125 amino acids of APP-SW.The MBP portion is captured on an assay substrate by anti-MBP captureantibody. Incubation of the captured fusion protein in the presence ofalpha-secretase and/or beta-secretase results in cleavage of thesubstrate at the alpha-secretase and/or beta-secretase cleavage sites,respectively. This system can be used to screen for the inhibitoryactivity of the agent(s) of interest. Analysis of the cleavage activitycan be, for example, by immunoassay of cleavage products. One suchimmunoassay detects a unique epitope exposed at the carboxy terminus ofthe cleaved fusion protein, for example, using the antibody SW192. Thisassay is described, for example, in U.S. Pat. No. 5,942,400.

Cellular Assays

Numerous cell-based assays can be used to evaluate the activity ofagent(s) of interest on relative alpha-secretase activity tobeta-secretase activity and/or processing of APP to releaseamyloidogenic versus non-amyloidogenic Aβ oligomers. Contact of an APPsubstrate with an alpha-secretase and/or beta-secretase enzyme withinthe cell and in the presence or absence of the agent(s) can be used todemonstrate alpha-secretase promoting and/or beta-secretase inhibitoryactivity of the agent( ). Preferably, the assay in the presence of theagent(s) provides at least about 30%, most preferably at least about 50%inhibition of the enzymatic activity, as compared with a non-inhibitedcontrol.

In one embodiment, cells that naturally express alpha-secretase and/orbeta-secretase are used. Alternatively, cells are modified to express arecombinant alpha-secretase and/or beta-secretase or synthetic variantenzymes, as discussed above. The APP substrate may be added to theculture medium and is preferably expressed in the cells. Cells thatnaturally express APP, variant or mutant forms of APP, or cellstransformed to express an isoform of APP, mutant or variant APP,recombinant or synthetic APP, APP fragment, or synthetic APP peptide orfusion protein containing the alpha-secretase and/or beta-secretase APPcleavage sites can be used, provided that the expressed APP is permittedto contact the enzyme and enzymatic cleavage activity can be analyzed.

Human cell lines that normally process Aβ from APP provide a usefulmeans to assay inhibitory activities of the agent(s). Production andrelease of Aβ and/or other cleavage products into the culture medium canbe measured, for example by immunoassay, such as Western blot orenzyme-linked immunoassay (EIA) such as by ELISA.

Cells expressing an APP substrate and an active alpha-secretase and/orbeta-secretase can be incubated in the presence of the agents todemonstrate relative enzymatic activity of the alpha-secretase and/orbeta-secretase as compared with a control. Relative activity of thealpha-secretase to the beta-secretase can be measured by analysis of oneor more cleavage products of the APP substrate. For example, inhibitionof beta-secretase activity against the substrate APP would be expectedto decrease release of specific beta-secretase induced APP cleavageproducts such as Aβ, sAPPβ and APPneo. Promotion or enhancement ofalpha-secretase activity against the substrate APP would be expected toincrease release of specific alpha-secretase induced APP cleavageproducts such as sAPPα and p3 peptide.

Although both neural and non-neural cells process and release Aβ, levelsof endogenous beta-secretase activity are low and often difficult todetect by EIA. The use of cell types known to have enhancedbeta-secretase activity, enhanced processing of APP to Aβ, and/orenhanced production of Aβ are therefore preferred. For example,transfection of cells with the Swedish Mutant form of APP (APP-SW); withthe Indiana Mutant form (APP-IN); or with APP-SW-IN provides cellshaving enhanced beta-secretase activity and producing amounts of Aβ thatcan be readily measured.

In such assays, for example, the cells expressing APP, alpha-secretaseand/or beta-secretase are incubated in a culture medium under conditionssuitable for alpha-secretase and/or beta-secretase enzymatic activity atits cleavage site on the APP substrate. On exposure of the cells to theagent(s), the amount of Aβ released into the medium and/or the amount ofCTF99 fragments of APP in the cell lysates is reduced as compared withthe control. The cleavage products of APP can be analyzed, for example,by immune reactions with specific antibodies, as discussed above.

Preferred cells for analysis of alpha-secretase and/or beta-secretaseactivity include primary human neuronal cells, primary transgenic animalneuronal cells where the transgene is APP, and other cells such as thoseof a stable 293 cell line expressing APP, for example, APP-SW.

In Vivo Assays: Animal Models

Various animal models can be used to analyze the activity of agent(s) ofinterest on relative alpha-secretase and/or beta-secretase activityand/or processing of APP to release Aβ. For example, transgenic animalsexpressing APP substrate, alpha-secretase and/or beta-secretase enzymecan be used to demonstrate inhibitory activity of the agent(s). Certaintransgenic animal models have been described, for example, in U.S. Pat.Nos. 5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015,and 5,811,633, and in Ganes et al., 1995, Nature 373:523. Preferred areanimals that exhibit characteristics associated with the pathophysiologyof AD. Administration of the agent(s) to the transgenic mice describedherein provides an alternative method for demonstrating the inhibitoryactivity of the agent(s). Administration of the agent(s) in apharmaceutically effective carrier and via an administrative route thatreaches the target tissue in an appropriate therapeutic amount is alsopreferred.

Inhibition of beta-secretase mediated cleavage of APP at thebeta-secretase cleavage site and of Aβ release can be analyzed in theseanimals by measure of cleavage fragments in the animal's body fluidssuch as cerebral fluid or tissues. Likewise, promotion or enhancement ofalpha-secretase mediated cleavage of APP at the alpha-secretase cleavagesite and of release of sAPPα can be analyzed in these animals by measureof cleavage fragments in the animal's body fluids such as cerebral fluidor tissues. In certain embodiments, analysis of brain tissues for Aβdeposits or plaques is preferred.

On contacting an APP substrate with an alpha-secretase and/orbeta-secretase enzyme in the presence of the agent(s) under conditionssufficient to permit enzymatic mediated cleavage of APP and/or releaseof Aβ from the substrate, desirable agent(s) are effective to reducebeta-secretase-mediated cleavage of APP at the beta-secretase cleavagesite and/or effective to reduce released amounts of Aβ. The agent(s) arealso preferably effective to enhance alpha-secretase-mediated cleavageof APP at the alpha-secretase cleavage site and to increase releasedamounts of sAPPα. Where such contacting is the administration of theagent(s) to an animal model, for example, as described above, theagent(s) is effective to reduce Aβ deposition in brain tissues of theanimal, and to reduce the number and/or size of beta amyloid plaques.Where such administration is to a human subject, the agent(s) iseffective to inhibit or slow the progression of disease characterized byenhanced amounts of Aβ, to slow the progression of AD in the, and/or toprevent onset or development of AD in a patient at risk for the disease.

Methods of Monitoring Clinical Efficacy

In various embodiments, the effectiveness of treatment can be determinedby comparing a baseline measure of a parameter of disease beforeadministration of the agent(s) (e.g., ASBIs such as galangin, rutin, andanalogues, derivatives, or prodrugs thereof) is commenced to the sameparameter one or more time points after the agent(s) or analog has beenadministered. One illustrative parameter that can be measured is abiomarker (e.g., a peptide oligomer) of APP processing. Such biomarkersinclude, but are not limited to increased levels of sAPPα, p3 (Aβ17-42or Aβ17-40), sAPPβ, soluble Aβ40, and/or soluble Aβ42 in the blood,plasma, serum, urine, mucous or cerebrospinal fluid (CSF). Detection ofincreased levels of sAPPα and/or p3, and decreased levels of sAPPβand/or APPneo is an indicator that the treatment is effective.Conversely, detection of decreased levels of sAPPα and/or p3, and/orincreased levels of sAPPβ, APPneo, Tau or phospho-Tau (pTau) is anindicator that the treatment is not effective.

Another parameter to determine effectiveness of treatment is the levelof amyloid plaque deposits in the brain. Amyloid plaques can bedetermined using any method known in the art, e.g., as determined by CT,PET, PIB-PET and/or MRI. Administration of the agent(s) (e.g., ASBIssuch as galangin, rutin, and analogues, derivatives, or prodrugsthereof) can result in a reduction in the rate of plaque formation, andeven a retraction or reduction of plaque deposits in the brain.Effectiveness of treatment can also be determined by observing astabilization and/or improvement of cognitive abilities of the subject.Cognitive abilities can be evaluated using any art-accepted method,including for example, Clinical Dementia Rating (CDR), the mini-mentalstate examination (MMSE) or Folstein test, evaluative criteria listed inthe DSM-IV (Diagnostic and Statistical Manual of Mental Disorders,Fourth Edition) or DSM-V, and the like.

Clinical efficacy can be monitored using any method known in the art.Measurable biomarkers to monitor efficacy include, but are not limitedto, monitoring blood, plasma, serum, urine, mucous or cerebrospinalfluid (CSF) levels of sAPPα, sAPPβ, Aβ42, Aβ40, APPneo and p3 (e.g.,Aβ17-42 or Aβ17-40). Detection of increased levels of sAPPα and/or p3,and decreased levels of sAPPβ and/or APPneo are indicators that thetreatment or prevention regime is efficacious. Conversely, detection ofdecreased levels of sAPPα and/or p3, and increased levels of sAPPβand/or APPneo are indicators that the treatment or prevention regime isnot efficacious. Other biomarkers include Tau and phospho-Tau (pTau).Detection of decreased levels of Tau and pTau are indicators that thetreatment or prevention regime is efficacious.

Efficacy can also be determined by measuring amyloid plaque load in thebrain. The treatment or prevention regime is considered efficacious whenthe amyloid plaque load in the brain does not increase or is reduced.Conversely, the treatment or prevention regime is consideredinefficacious when the amyloid plaque load in the brain increases.Amyloid plaque load can be determined using any method known in the art,e.g., including CT, PET, PIB-PET and/or MRI.

Efficacy can also be determined by measuring the cognitive abilities ofthe subject. Cognitive abilities can be measured using any method knownin the art. Illustrative tests include assigning a Clinical DementiaRating (CDR) score or applying the mini mental state examination (MMSE)(Folstein, et al., Journal of Psychiatric Research 12 (3): 189-98).Subjects who maintain the same score or who achieve an improved score,e.g., when applying the CDR or MMSE, indicate that the treatment orprevention regime is efficacious. Conversely, subjects who receive ascore indicating diminished cognitive abilities, e.g., when applying theCDR or MMSE, indicate that the treatment or prevention regime has notbeen efficacious.

In certain embodiments, the monitoring methods can entail determining abaseline value of a measurable biomarker or parameter (e.g., amyloidplaque load or cognitive abilities) in a subject before administering adosage of the agent(s), and comparing this with a value for the samemeasurable biomarker or parameter after treatment.

In other methods, a control value (e.g., a mean and standard deviation)of the measurable biomarker or parameter is determined for a controlpopulation. In certain embodiments, the individuals in the controlpopulation have not received prior treatment and do not have AD, MCI,nor are at risk of developing AD or MCI. In such cases, if the value ofthe measurable biomarker or clinical parameter approaches the controlvalue, then treatment is considered efficacious. In other embodiments,the individuals in the control population have not received priortreatment and have been diagnosed with AD or MCI. In such cases, if thevalue of the measurable biomarker or clinical parameter approaches thecontrol value, then treatment is considered inefficacious.

In other methods, a subject who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for one ormore of the biomarkers or clinical parameters to determine whether aresumption of treatment is required. The measured value of one or moreof the biomarkers or clinical parameters in the subject can be comparedwith a value previously achieved in the subject after a previous courseof treatment. Alternatively, the value measured in the subject can becompared with a control value (mean plus standard deviation/ANOVA)determined in population of subjects after undergoing a course oftreatment. Alternatively, the measured value in the subject can becompared with a control value in populations of prophylactically treatedsubjects who remain free of symptoms of disease, or populations oftherapeutically treated subjects who show amelioration of diseasecharacteristics. In such cases, if the value of the measurable biomarkeror clinical parameter approaches the control value, then treatment isconsidered efficacious and need not be resumed. In all of these cases, asignificant difference relative to the control level (e.g., more than astandard deviation) is an indicator that treatment should be resumed inthe subject.

In certain embodiments the tissue sample for analysis is typicallyblood, plasma, serum, urine, mucous or cerebrospinal fluid from thesubject.

Kits.

In various embodiments, the active agent(s) (e.g., APP specific BACEinhibitor (ASBI) such as galangin, rutin, and analogues, derivatives, atautomer or stereoisomer thereof, or prodrug thereof as describedherein) can be enclosed in multiple or single dose containers. Theenclosed agent(s) can be provided in kits, for example, includingcomponent parts that can be assembled for use. For example, an activeagent in lyophilized form and a suitable diluent may be provided asseparated components for combination prior to use. A kit may include anactive agent and a second therapeutic agent for co-administration. Theactive agent and second therapeutic agent may be provided as separatecomponent parts. A kit may include a plurality of containers, eachcontainer holding one or more unit dose of the compounds. The containersare preferably adapted for the desired mode of administration,including, but not limited to tablets, gel capsules, sustained-releasecapsules, and the like for oral administration; depot products,pre-filled syringes, ampules, vials, and the like for parenteraladministration; and patches, medipads, creams, and the like for topicaladministration, e.g., as described herein.

In certain embodiments, a kit is provided where the kit comprises: oneor more ASBI compounds described herein, or prodrug, a tautomer orstereoisomer thereof, or pharmaceutically acceptable salt or solvate ofsaid compound, said stereoisomer, or said tautomer or prodrug preferablyprovided as a pharmaceutical composition and in a suitable container orcontainers and/or with suitable packaging; optionally one or moreadditional active agents, which if present are preferably provided as apharmaceutical composition and in a suitable container or containersand/or with suitable packaging; and optionally instructions for use, forexample written instructions on how to administer the compound orcompositions.

In another embodiment, a kit is provided that comprises a singlecontainer or multiple containers: (a) a pharmaceutically acceptablecomposition comprising one or more ASBI compounds described and/orclaimed herein, or a tautomer or stereoisomer thereof, orpharmaceutically acceptable salt or solvate of said compound, saidstereoisomer, or said tautomer, optionally a pharmaceutically acceptablecomposition comprising one or more additional therapeutic agents; andoptionally instructions for use their use. The kit may optionallycomprise labeling (e.g., instructional materials) appropriate to theintended use or uses.

As with any pharmaceutical product, the packaging material(s) and/orcontainer(s) are designed to protect the stability of the product duringstorage and shipment. In addition, the kits can include instructions foruse or other informational material that can advise the user such as,for example, a physician, technician or patient, regarding how toproperly administer the composition(s) as prophylactic, therapeutic, orameliorative treatment of the disease of concern. In some embodiments,instructions can indicate or suggest a dosing regimen that includes, butis not limited to, actual doses and monitoring procedures.

In some embodiments, the instructions can include informational materialindicating that the administering of the compositions can result inadverse reactions including but not limited to allergic reactions suchas, for example, anaphylaxis. The informational material can indicatethat allergic reactions may exhibit only as mild pruritic rashes or maybe severe and include erythroderma, vasculitis, anaphylaxis,Steven-Johnson syndrome, and the like. In certain embodiments theinformational material(s) may indicate that anaphylaxis can be fatal andmay occur when any foreign protein is introduced into the body. Incertain embodiments the informational material may indicate that theseallergic reactions can manifest themselves as urticaria or a rash anddevelop into lethal systemic reactions and can occur soon after exposuresuch as, for example, within 10 minutes. The informational material canfurther indicate that an allergic reaction may cause a subject toexperience paresthesia, hypotension, laryngeal edema, mental statuschanges, facial or pharyngeal angioedema, airway obstruction,bronchospasm, urticaria and pruritus, serum sickness, arthritis,allergic nephritis, glomerulonephritis, temporal arthritis,eosinophilia, or a combination thereof.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

In some embodiments, the kits can comprise one or more packagingmaterials such as, for example, a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (I.V.) bag, envelope, and the like;and at least one unit dosage form of an agent comprising active agent(s)described herein and a packaging material. In some embodiments, the kitsalso include instructions for using the composition as prophylactic,therapeutic, or ameliorative treatment for the disease of concern.

In some embodiments, the kits can comprise one or more packagingmaterials such as, for example, a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (I.V.) bag, envelope, and the like;and a first composition comprising at least one unit dosage form of anagent comprising one or more active agent(s) (e.g., APP specific BACEinhibitor (ASBI) such as galangin, rutin, and analogues, derivatives, atautomer or stereoisomer thereof, or prodrug thereof as describedherein) within the packaging material, along with a second compositioncomprising a second agent such as, for example, an agent used in thetreatment and/or prophylaxis of Alzheimer's disease (e.g., as describedherein), or any prodrugs, codrugs, metabolites, analogs, homologues,congeners, derivatives, salts and combinations thereof. In someembodiments, the kits may also include instructions for using thecomposition as a prophylactic, therapeutic, or ameliorative treatmentfor the disease of concern.

In certain embodiments the instructions/instructional materials whenpresent teach dosages and/or treatment regimen(s) and/orcounter-indictions for the active agents contained in the kit. While theinstructional materials, when present, typically comprise written orprinted materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 APP-Specific BACE Inhibitors (ASBI) A Novel Class ofTherapeutic Agents for Alzheimer's Disease

A critical limitation of protease inhibitory strategies (e.g., BACEinhibitors) in the treatment of various pathologies is the promiscuityof substrate target effects, i.e., the inhibition of cleavage of allsubstrates of a given targeted protease, such as BACE or the γ-secretasecomplex. In the case of γ-secretase, substrates other than APP, such asNotch, raise concerns for potential side effects of γ-secretaseinhibition, and the recent failure of the γ-secretase inhibitor,Semagacestat, serves to reinforce such concerns. In the case of BACE,inhibition of non-APP substrates such as PSGL1 or LRP raises similarconcerns.

Therefore, the optimal BACE inhibitor would be one that would bind notto BACE but rather to APP, leading to APP-specific BACE inhibition(ASBI). Such a therapeutic would represent a new class of Alzheimer'sdisease therapeutics: ASBIs.

The data reported in this example on the identification of the firstASBIs demonstrate that such an approach is feasible. APP-specific BACEinhibitors (ASBIs) inhibit the BACE cleavage of the amyloid precursorprotein (APP) but not the proteolytic cleavage of other substrates.Through screening of a small library of 448 clinical compounds (NCC3364) in the ASBI assays a bioflavonoid was identified—rutin, a citrusflavonoid glycoside—that was effective as an ASBI at a dose of 1 μM.Further analysis of a group of bioflavonoids revealed a second molecule,galangin, a bioflavonoid from the galangal rhizome, that similarly actsas an ASBI at a dose of 50 μM. These bioflavonoids represent the firstmembers of what believed to be new class of disease-modifyingtherapeutic agents for AD.

It is believed, the systematic application of the approach outlinedherein to identify APP-specific BACE inhibitors, and evaluate their invivo ability to modulate APP processing specifically, has not beenpreviously reported. A bioflavonoid nutritional supplement is identifiedthat provides molecular lead compound that acts as an ASBI in cellmodels. It has been shown that increasing brain levels of thisbioflavonoid through a prodrug approach leads to reduction of Aβ42 uponin the AD mouse model. Thus ASBIs represent a novel class of therapeuticagents for AD.

Materials and Methods

Compounds.

Rutin (ASBI-1) was obtained from Sigma (cat # R5143, St. Louis, Mo.),Galangin (ASBI-2) was obtained from Sigma (cat#282200, St. Louis, Mo.)and progalangin (PG-1) was synthesized.

ASBI Assay.

Consist of two parts a) evaluation of a compound for inhibition of theBACE cleavage of the MBP-C125 substrate and b) evaluation of thecandidate for BACE inhibition of the P5-P5′ substrate.

a) MBP-C125 Cleavage Assay.

A protein construct of Maltose-Binding-Protein carrying the 125C-terminal residues of APP (1 uL of 1 mM in water) was incubated with aflavonoid (100 uM) for 15 minutes. The mix was then submitted tocleavage by BACE (Sigma #B9059, 5 μl of 3 unit/ml in BACE buffer) for 30minutes. After 0, 10, 20 and 30 minutes, 2 uL the reaction mixture werefrozen and the different time points were quantified simultaneously forthe amount of APP-C99 created. The quantification was done by using aPerkin Elmer ALPHALisa Amyloid Amyloid Beta kit (AL275C) modified byreplacing the anti-Abeta acceptor beads (AL275AC) with anti-APP acceptorbeads (AL275AC) and making the antibody mix with 2×ALPHALisa buffer.

b) P5-P5′ Cleavage Assay.

The inhibition of BACE cleavage of the fluorescence substrate P5-P5′ bythe flavonoids at 100 uM was measured using the β-Secretase (BACE1)Activity Detection Kit from Sigma-Aldrich (CS0010) and the standardprotocol.

Plasmids.

The pAPtag5-NRG1-β1 construct was kindly provided by Dr. Carl Blobel(Horiuchi et al. (2005) Dev. Biol. 283: 459-471). The BACE1 constructwas a gift from Dr. Michael Willem and Dr. Christian Haass (Willem etal. (2006) Science 314: 664-666). Constructs pCMV5-Mint3, pMst-AβPP,pG5E1B-luc, and pCMV-LacZ were generously provided by Dr. Thomas Südhof,Dr. Patrick Mehlen, and Dr. Veronique Corset (Cao (2001) Science 293:115-120. Construct pEF-N-FLAG-TAZ was kindly provided by Dr. MichaelYaffe and Dr. lain Farrance. Construct pcDNA3.1-APLP2-Gal4 was describedpreviously (Orcholski et al. (2011) J. Alzheimers Dis. 23: 689-699).

Cell Culture and Western Blot.

The Chinese Hamster Ovary (CHO) cell line over-expressing human AβPP (7W) was kindly provided by Dr. Edward Koo. Plasmid constructs weretransiently transfected into HEK293T or 7 W cells with Lipofectamine2000 (Invitrogen). Western Blot analysis was performed as previouslydescribed (Swistowski et al. (2009) J. Neurosci., 29: 15703-15712).Briefly, 48 hr after transfection, cells were harvested and lysed inNP-40 Cell Lysis Buffer (50 mM TrisHCl, pH 8.0, 150 mM NaCl and 1%NP-40). Cell lysates were mixed with 1×LDS loading buffer (Invitrogen)and 50 mM DTT, and boiled at 100° C. for 10 min. After SDS-PAGE andelectrotransfer, Western blotting was performed using anti-APPantibodies (CT15, a kind gift from Dr. Edward Koo, for β-CTF; 6E10(Covance) for full length APP and sAPPα). Thirty minutes of TBS-Tweenwash were followed by incubation with secondary antibodies.

Neuregulin1 Shedding Assay.

A cDNA construct encoding a human placental secreted alkalinephosphatase (SEAP)-NRG1 (pAPtag5-NRG1-β1) fusion protein was transfectedin HEK293 cells in a 6-well format with or without full-length wild-typeBACE1 using Lipofectamine 2000 (Invitrogen) as described previously(Vassar et al. (1999) Science, 286: 735-741). After transfection themedium was replaced with DMEM containing 10% heat-inactivated FetalBovine Serum and incubated for 24 hr. SEAP activity was measured in theconditioned medium. For alkaline phosphatase activity measurements, 200μl of reaction solution (0.1 M glycine, pH 10.4, 1 mM MgCl₂, 1 mM ZnCl₂containing 1 mg/ml 4-nitrophenyl phosphate disodium salt hexahydrate,Sigma) were added to 20 μl of the conditioned medium. The absorbance wasread at 405 nm. Statistical analyses were performed using a two tailedStudent's t-test.

Transactivation Assay.

HEK293T cells were co-transfected with five plasmids: (1) pG5E1B-luc,0.3 μg; (2) pCMV-LacZ, 0.1 μg; (3) pMst-APP (APP-Gal4) orpcDNA3.1-APLP2-Gal4, 0.3 μg; (4) pCMV5-Mint3, 1.0 μg; (5)pEF-N-FLAG-TAZ, 1.0 μg. Cells were harvested 48 hr after transfection in0.2 ml per well Cell Culture Lysis Buffer (Promega), and theirluciferase and β-galactosidase activities were determined with thePromega luciferase assay kit and the Promega β-galactosidase assay kit,respectively. The luciferase activity was standardized by theβ-galactosidase activity to control for transfection efficiency andgeneral effects on transcription. Transfections were performed at 80-90%cofluency in six-well plates using Lipofectamine 2000 (Invitrogen).

Surface Plamon Resonance (SPR) Testing.

The surfaces of four flow cells (FC1, FC2, FC3, FC4) of acarboxymethylated-dextran (CM-5) chip were washed sequentially with 50mM NaOH, 1 mM HCl, 0.05% H₃PO₄ and 20 mM sodium phosphate pH 7.4, 125 mMsodium chloride in parallel using a flow rate of 30 μl/min for 1 minusing a Biacore T-100 (GE Healthcare). Three fusion proteins wereimmobilized via amine coupling using 20 mM phosphate, 125 mM sodiumchloride pH 7.4. The three proteins were MBP-eAPP₂₃₀₋₆₂₄—a fusionprotein containing maltose binding protein (MBP) and residues 230-624 ofthe ectodomain of APP (90-kDa) (FC4), eAPP₂₃₀₋₆₂₄—a protein thatcontains only residues 230-624 (45-kDa)(FC2), and TRX-eAPP₅₇₅₋₆₂₄—afusion protein containing thioredoxin (TRX) and residues 575-624 of theectodomain (20-kDa) (FC3). The proteins were produced as described inLibeu, et al. (2011) J. Alzheimers Dis., 25(3): 547-566 Libeu et al(2011). The flow cell FC1 was used as a control. Galangin was dilutedfrom 10 mM solutions in DMSO to 50 μM in 1% DMSO, 20 mM sodium phosphatepH 7.4, 125 mM sodium chloride, 0.05% Tween and then serially diluted by1.5 for 10 steps. Binding traces were recorded for each dilution with abinding phase of 60 seconds and a dissociation phase of 240 seconds.Each cycle was performed at 20° C. with a constant flow rate of 20μl/min. An additional 240 seconds of buffer flow at 60 μl per min acrossthe cells was applied as a regeneration phase to facilitate completedissociation of the compound from the protein. The sensograms wereobtained by subtraction of the reference and buffer signals using theBiacore T100 Evaluation software. The binding curves were modeled withthe PRISM (Graphpad Inc).

Pharmacokinetic (PK) Analysis.

The brain penetrance of galangin and progalangin were assessed in astandard PK comprising subcutaneous (Sub-Q) injection of 5 adultnon-transgenic mice with 50 ul of a 5 mg/ml stock of compound indimethylsulfoxide (DMSO) or a dose of 10 mg/kg for a 25 g mouse.Injected mice were anesthetized with ketamine/xylazine at 1, 2, 4, 6,and 8 hours and blood collected by cardiac puncture. The mice were thenperfused with saline and brain tissue dissected and snap frozen on dryice. Blood was centrifuged at 3000 rpm for 10 minutes and the plasmasupernatant collected. Both plasma and the right hemibrain were sent toIntegrated Analytical Solutions (IAS, Berkeley, Calif.) with a referencesample of compound for compound level analysis in tissue and plasma. Thecompound levels were determined using a LC-MS/MS approach.

Pilot Efficacy Testing.

Galangin and progalangin (PG-1) were dissolved in 10% solutol/15%DMSO/75% polyethylene glycol (PEG). Stock solutions were prepared at 10mg/ml for each compound and 100 ul was injected subcutaneously daily for14 days at a dose of 40 mkd. There were 5 PDAPP AD model J20 mice inboth the galangin and progalangin groups, and 9 vehicle-only treated J20controls. Mice were anesthetized, blood collected for plasma, and braintissue collected as described above 2 hours after injection on the lastday of treatment. The right hemibrain was further microdissected toisolate hippocampus and entorhinal cortex and this combined tissue wasused for biochemical analysis. The remaining tissue and plasma was sentto IAS for compound level analysis.

Biochemistry.

Aβ1-42 and Aβ1-40 levels were determined using thehippocampal/entorhinal cortical tissue. Briefly, frozen tissue sampleswere weighed, and a 20% w/v sonicate prepared in 5M guandine-HCl/50 mMTris, pH8. Sonication was performed with sample tubes in ice water, 4×5seconds at 60 Hz, then 3×5 seconds at 80 Hz. Samples were then rotatedat room temperature for 3 hours and frozen at −20° C. until assay.Invitrogen ELISA kits were used for Aβ1-40 and Aβ1-42 according to themanufacturer's instructions.

Results

Identification of APP-Specific BACE Inhibitors (ASBIs)

A primary high-throughput screening (HTS) assay was set up foridentification of ASBIs using a dual-substrate testing paradigm (FIGS.3A & 3B). The previously reported (Sinha et al. (1999) Nature, 402:537-540) BACE substrate the MBP-C125 APP₆₉₅ wt fusion protein consistingof maltose binding protein fused to the carboxyterminal 125 amino acidsof wild type APP, was used as the primary substrate; and thecommercially available P5-P5′ fluorescence substrate, derived from theP5-P5′ residues of the BACE cleavage site of APP, was used as thesecondary substrate. Each of these substrates was incubated withrecombinant BACE (R&D (cat#931-AS-050) in a 96-well plate format. Forthe MBP-C125 substrate, the C-99 product from the BACE cleavage wasmeasured using an AlphaLISA assay as a readout (FIG. 3B). For the P5-5′substrate, the loss of fluorescence following BACE cleavage was used asthe readout. An ASBI would be predicted to inhibit the BACE cleavage ofthe MBP-C125 substrate, while not necessarily inhibiting cleavage of thefluorescence substrate, depending on where the ASBI bound the APPsubstrate.

Based on the preliminary screening of a clinical compound library of 448compounds, the hit-rate was anticipated to be very low, since only onecompound was identified in the initial screen. Dose response curves ofpotential ‘hits’ were next be done to identify validated ‘hits’ forfurther development. The HTS screening was performed at an initialconcentration of 10 uM for each candidate. An initial screen of a smallclinical compound library of 448 commercially available clinicalcompounds was completed. The screen yielded a single hit (FIG. 3A),identified as the bioflavonoid rutin (ASBI-1, also referred to asrutoside), which is derived from the citrus flavonoid glycoside found inbuckwheat. This bioflavonoid decreased sAPPβ in SH-SY5Y cells, and wasshown to be specific for APP, supporting the notion that rutin actsspecifically on the BACE cleavage of APP. Next a panel of bioflavonoids(FIG. 4) was tested, first for their abilities to inhibit sAPPβ in thecells. Another bioflavonoid, galangin, was identified that also behavedas an ASBI, inhibiting the cleavage of the MBP substrate by BACE (FIG.4, diamonds) while showing no inhibition of the P5-P5′ substrate (FIG.4, circles). Galangin (ASBI-2) is a flavanol found in galangal rhizome,and is commonly used as a nutritional supplement.

Effect of Bioflavonoid ASBIs on sAPPβ and APP Processing in Cells.

APP is processed through two major pathways: the non-amyloidogenicpathway involves α-secretase cleavage, proteolyzing APP into sAPPα andα-CTF (C83), while the amyloidogenic pathway starts with β-secretasecleavage, cleaving APP into sAPPβ and β-CTF (C99). The β-CTF is thencleaved by the γ-secretase, which produces Aβ and AICD. The ability ofthe ASBIs to inhibit the β-secretase processing of APP was tested inSH-SY5Y neuroblastoma cells that express APP. The sAPPβ fragment formedfrom the BACE cleavage product was measured using an AlphaLisa assayfrom Perkin-Elmer (Cat# A2132). Following the discovery of rutin in theinitial screen, it was demonstrated that at 1 μM it slightly inhibitedthe production of sAPPβ by SH-SY5Y cells (FIG. 5A). Testing of a panelof bioflavonoids led to the identification of galangin as an ASBI.Treatment of SH-SY5Y cells with galangin similarly decreased sAPPβlevels at 50 μM. No effect on APP levels was detected.

Bioflavonoid ASBIs Inhibit APP-Gal4 and APLP2-Gal4 Transactivation

While the APP-C31 cleavage is associated with cell death, the APPintracellular domain (AICD) created following the γ-secretase cleavagehas been implicated in various signaling pathways, and has been shown tomodulate the expression of many genes including KAI1, neprilysin, andAPP itself (Hong et al. (2000) Science, 290: 150-153). AnAPP-Gal4/Mint3/TAZ transactivation assay (Maillard et al. (2007) J. Med.Chem., 50: 776-781; Hardy et al. (1991) Trends Pharmacol. Sci., 12:383-388) was established, and using this assay, it was found that ASBIsinhibited APP-Gal4 transactivation (FIG. 7). To confirm this effect, theAPP-Gal4/Fe65 transactivation assay was employed. The effect of theASBIs in the APLP2-Gal4 transactivation was examined (FIG. 7). Theseresults indicate that rutin (ASBI-1) and galangin (ASBI-2) inhibit bothAPP and related family member APLP2-Gal4 transactivation.

Interaction of ASBIs with APP

To explore the interaction of ASBIs with APP a ligand blot technique wasused, where MBP-C125 APP was dot blotted on a nitrocellulose blot andbinding to the protein was detected upon treatment with thebioflavonoids. A nitrocellulose filter-binding assay with bovine serumalbumin (BSA) was used as a control. The binding of bioflavonoids wasdetermined using both UV and MALDI mass spectrometric analysis. This isa qualitative measure of protein small molecule interaction but doesshow that the ASBI bound to APP.

Surface plasmon resonance (SPR) was then used to demonstrate binding toAPP and to determine the affinity of galangin for APP.

Surface Plasmon Resonance (SPR) Screening:

The binding affinity of the compounds for the ectodomain of APP wasdetermined using SPR. A technique for measuring the affinity ofcompounds to fragments of the ectodomain of APP was developed. For thegalangin binding experiments TRX-eAPP575-624 was used. The eAPP wascrosslinked linked to the CM5 Biacore chips (GE Healthcare). Galangin atvarious concentrations were used in the flow through over the chip andthe plasmon resonance signal was determined using a Biacore T100 (FIG.5B).

Bioflavonoid ASBI Treatment Reduces Aβ in an AD Transgenic Mouse Model

The brain permeability of the bioflavonoids rutin and galangin in micewas evaluated and it was found that after a 10 mpk sc administration norutin was detectable in the brain, while low levels of galangin (Cmax˜50ng/g at 1 h) could be detected in the brain (FIG. 8). The brain toplasma ratio was 1:10. In order to see if the brain levels of galangincould be enhanced, the prodrug (PG-1) was tested and it resulted inincreased delivery of galangin to the brain (Cmax˜100 ng/g at 1 h).Based on these pharmacokinetic analysis it was decided to test galanginand progalangin (PG-1) in an AD mouse model, the PDAPP (J20) mice.

Treatment of J20 mice at 40 mpk with galangin shows some reduction ofAβ40 while Aβ42 was unchanged in the hippocampus and the cortex.However, treatment with progalangin shows both reduction of Aβ40 andAβ42 consistent with the increased brain levels of galangin seen upontreatment with the prodrug. These results, taken together, indicate thatthe bioflavonoid galangin interacts directly with APP, inhibits BACEcleavage of APP but not neuregulin or a BACE-target peptide, inhibitsBACE-dependent APP nuclear signaling, and reduces Aβ1-42 in a transgenicmouse model of AD.

Discussion

Two bioflavonoid analogs that are used as nutritional supplements andthat inhibit the β-secretase mediated APP processing by a novelmechanism were identified. These molecules inhibit the BACE cleavage ofthe MBP-C125 APP substrate, resulting in the inhibition of theproduction of C99, but do not inhibit cleavage of the β-site peptidesubstrate (P5-P5′). In addition, these bioflavonoids reduce sAPPβ inneuroblastoma SH-SY5Y cells, whereas galangin fails to reduce neuregulinBACE-dependent shedding. Further, it was demonstrated that the activityis associated with binding to the MBP-C125 substrate. These findingsdefine a new mechanism to modulate APP processing.

The approach described herein addresses a critical limitation of theprotease inhibitory strategies for Alzheimer's disease (AD), providing amechanism by which the inhibition of cleavage of all substrates of agiven targeted protease, such as BACE or the γ-secretase complex, isavoided. The γ-secretase substrates other than APP, such as Notch, raiseconcerns for potential side effects of γ-secretase inhibition, and therecent failure of the γ-secretase inhibitor, Semagacestat, serves toreinforce such concerns. In the case of BACE, non-APP substrates such asPSGL1 and LRP raise similar concerns. Therefore, the optimal BACEinhibitor would be one that would bind to APP rather than to BACE,leading to APP-specific BACE inhibition (ASBI). Such a therapeutic, asdescribed herein, represents a new class of Alzheimer's diseasetherapeutics.

Two known BACE substrates are likely to be important in immunologicalfunction: the P-selectin glycoprotein ligand-1 (Lichtenthaler et al.(2003) J. Biol. Chem. 278: 48713-48719), which mediates leukocyteadhesion, and the sialyl-transferase ST6Gal I (Kitazume et al. (2003) J.Biol. Chem., 278: 14865-14871), an enzyme that is secreted aftercleavage and is involved in regulating immune responses. The interactionof a sialyl-alpha-2,6 galactose residue, which is synthesized solely byST6Gal I, with a B-cell-specific lectin, CD22/Siglec-2, is important forB-cell function (Id.). It is notable that mice deficient in someglycosylation enzymes appear to grow normally but show subtleneurological abnormalities with increasing age;glycosphingolipid-deficient mice show lethal audiogenic seizures inducedby a sound stimulus. In this regard, it is important to note that noreports have yet appeared on the response of BACE1 null mice to immunechallenge. BACE1 has also been shown to process the APP homologue,APLP2; this homologue has a different sequence specificity than that ofAPP around the putative BACE1 cleavage site, yet galangin also inhibitedcleavage of APLP2 by BACE. The levels of APLP2 proteolytic products weredecreased in BACE1 deficient mice and increased in BACE1 overexpressingmice (Pastorino et al. (2004) Mol. Cell Neurosci., 25: 642-649). Giventhe great need for disease modifying therapies in AD, this approach ofdeveloping a APP substrate-specific BACE inhibitor is novel and couldlead to clinical candidates that are effective against the disease.

An HTS assay to identify ASBIs was set up. Initial screening of aclinical library of 448 compounds in this assay led to theidentification of a bioflavonoid that specifically inhibited theMBP-C125 substrate of BACE while not preventing the cleavage of theP5-P5′ substrate. This bioflavonoid, rutin, is a nutritional supplementthat was also found to inhibit sAPPβ production in cells. A panel ofbioflavonoids was then tested in the ASBI and sAPPβ assays in cellculture. From this testing another bioflavonoid, galangin, wasidentified. Galangin is another nutritional supplement that waseffective in the ASBI assay, as well as in cells, in preventing the BACEcleavage of APP. Using a simple nitrocellulose filter ligand-bindingassay initial binding of various bioflavonoids to the MBP-C125 substratewas demonstrated. A panel of bioflavonoids was screened in the ASBIassay. However, only rutin and galangin were effective as ASBIs (FIG.4). Galangin modulates sAPPβ levels in cells and demonstrated binding tothe APP substrate (FIG. 5). Interestingly, galangin also has beenreported to be an inhibitor of acetylcholine esterase (AChE) (Guo et al.(2010) Chemico-Biol. Interaction, 187: 246-248) and to induce autophagy(Wen et al. (2012) Pharmacology, 89: 247-255).

It was demonstrated that the bioflavonoids inhibit BACE cleavage of APPand APLP2, using a HEK-293 assay transfected with APP or APLP2-Gal4(see, e.g., Orcholski et al. (2011) J. Alzheimers Dis., 23(4):689-99 fora description of the assay). Transactivation is achieved upontransfection with Mint3 and Taz. The ASBI as expected to inhibit onlythe transactivation of APP-Gal4, not that of APLP2-Gal4. However,galangin inhibited both APP-Gal4 and APLP2-Gal4. Thus galangin exhibitsAPP-family specificity rather than APP specificity; however, given thedemonstration of Aβ-like fragments derived from APLP2, the ability toinhibit BACE cleavage of both APP and APLP2 may be more desirable thaninhibiting the cleavage of APP alone (Eggert et al. (2004) J. Biol.Chem. 279(18): 18146-18156).

Initial pharmacokinetic evaluation of these two bioflavonoids in brainuptake assays using NTg mice showed that rutin does not cross theblood-brain barrier, whereas galangin did show some brain penetration,thus enabling its evaluation for proof-of-concept studies in thetransgenic (Tg) mouse model. Galangin was then evaluated for its in vivoeffect on Aβ40 and Aβ42 (FIG. 7). The reduction of Aβ levels is veryencouraging in this study. Further increase in brain levels of galanginis possible using a prodrug of galangin (PG-1), and it was demonstratedthat PG-1 is more effective in vivo than galangin to reduce Aβ40 andAβ42.

In conclusion, this current study indicates that certain bioflavonoidshave the ability to bind APP and inhibit the BACE cleavage of APP andAPLP2 thus suggesting that they function as APP specific BACEinhibitors. These represent a new class of therapeutics for Alzheimer'sdisease that would be devoid of the potential toxicity from directinhibition of BACE. Galangin as its prodrug analog, progalangin-1 wasalso shown to be effective in reducing Aβ40 and Aβ42 in the AD mousemodel.

Example 2 Progalangin as an ASBI

CNS exposure studies were performed and consisted of a time-coursedesign to collect heparinized plasma and brains. Following scadministration of the galangin or progalangin (compound-2) at 10 mg/kg,plasma and brain levels of the compounds were determined by quantitativeLC/MS/MS methodology. Plasma samples were precipitated withacetonitrile:methanol (1:1) cocktail containing an internal standard.The brain samples were homogenized directly in ethylacetate or extractedfrom 5M guanidine homogenates with the liquid-liquid method. Theresulting supernatant was evaporated to dryness and subjected to theLC/MS/MS analysis. For each compound 5 mice were used for the analysis.The brain-to-plasma ratios and plasma/brain Cmax levels were thendetermined (see, e.g., FIG. 8).

Experimental Procedures—Compound Synthesis.

5,7-Diacetoxyflavone

5,7-Dihydroxy-2-phenyl-4H-chromen-4-one (5.00 g, 19.67 mmol) was addedto a solution of acetic anhydride in pyridine (1:5, 42 mL) and thereaction mixture was stirred for 60 hours at ambient temperature. Thereaction mixture was diluted with diethyl ether (100 mL) and filtered.The solids were washed with additional diethyl ether (3×50 mL) and driedunder high vacuum to afford 4-oxo-2-phenyl-4H-chromene-5,7-diyldiacetate (6.40 g, 18.92 mmol, 96%) as a white crystalline solid.

¹H NMR (400 MHz, CDCl₃) δ 2.36 (s, 3H, CH₃COO), 2.45 (s, 3H, CH₃COO),6.67 (s, 1H, H-3), 6.85 (d, J=2 Hz, 1H, H-6), 7.36 (d, J=2 Hz, 1H, H-8),7.50 (m, 3H, H-3′, 5′, H-4′), 7.84 (dd, J=8 Hz, 2H, H-2′, 6′);

¹³C NMR (100 MHz, CDCl₃) δ 21.2 (q, CH₃COO), 21.3 (q, CH₃—C═O), 108.7(d, C-3), 109.1 (d, C-8), 113.7 (d, C-6), 115.1 (s, C-4a), 126.3 (2×d,C-2′, 6′), 129.2 (2×d, C-3′, 5′), 131.2 (d, C-4′), 131.9 (s, C-1′),150.3 (s, C-5), 154.0 (s, C-7), 157.8 (s, C-8a), 162.6 (s, C-2), 168.1(s, CH₃—C═O), 169.5 (s, CH₃COO), 176.5 (s, C-4).

Preparation of DMDO:

A 3 L, three-necked, round-bottomed reaction flask was equipped with anefficient mechanical stirrer, an addition funnel for solids, and acondenser (30 cm), set for downward displacement, attached to a twonecked receiving flask, the latter cooled at −78° C. by means of a dryice/acetone bath. The reaction flask was charged with a mixture of water(254 mL), acetone (192 mL), and NaHCO3 (58 g) and cooled at 5-10° C.with the help of an ice/water bath. While vigorously stirring andcooling, solid OXONE® (120 g, 0.195 mol) was added in five portions at 3minute intervals. After 3 min of the last addition, the addition funnelwas replaced with a stopper, and a moderate vacuum (80-100 mmHg) wasapplied to the flask. The cooling bath (5-10° C.) was removed from thereaction flask, and while vigorously stirring the DMDO/acetone solutionwas distilled and collected in the cooled (−78° C.) receiving flask overa period of 90 minutes. The receiving flask was warmed to −20° C. anddried for 3 hours over K₂CO₃. The DMDO solution was filtered into a dryflask and kept at −20° C. until used. Approximately 130 ml of DMDOsolution was collected. The concentration of DMDO was determined by NMRby dissolving a 0.2 mL aliquot of the dried DMDO solution in CDCl₃ andcomparing the height of the methyl proton signal of thedimethyldioxirane (at δ 1.65) with that of the ¹³C satellite peak to theright of acetone (0.5%), resulting in a 0.05 M DMDO solution. Thisanalysis must be done without delay!

5,7-Diacetoxy-3-hydroxyflavone

4-Oxo-2-phenyl-4H-chromene-5,7-diyl diacetate (1.80 g, 5.32 mmol) wasadded to a suspension of dried powdered 4 Å molecular sieves (1.80 g) inDCM (32 ml) and cooled to 0° C. DMDO solution (120 ml, 7.02 mmol, 1.32equiv.) was added dropwise. The resulting solution was stirred at 0° C.for 3 hours and allowed to warm to ambient temperature and stirred atthat temperature for 48 hours. The reaction mixture was filtered througha layer of anhydrous sodium sulfate on a bed of CELITE® and thevolatiles were removed in vacuo at ambient temperature to afford crude7-oxo-1α-phenyl-7,7α-dihydro-1αH-oxireno[2,3-b]chromene-4,6-diyldiacetate (ca. 1.90 g) as an oil. This oil was carried through to thenext stage.

Crude reaction mixture (ca. 1.90 g) was stirred in DCM (32 ml)containing 15 mg of p-TSA. The reaction mixture solidified almostimmediately. The reaction mixture was diluted with DCM (20 ml) andstirred for 2 days where TLC analysis indicated consumption of7-oxo-1α-phenyl-7,7α-dihydro-1αH-oxireno[2,3-b]chromene-4,6-diyldiacetate. The reaction mixture was adsorbed onto silica gel andrepeated flash column chromatography (Chloroform as eluent) affordimpure 3-hydroxy-4-oxo-2-phenyl-4H-chromene-5,7-diyl diacetate (800 mg).Further purification was achieved by recrystallization fromacetone/diethyl ether mixtures to afford3-hydroxy-4-oxo-2-phenyl-4H-chromene-5,7-diyl diacetate (660 mg, 1.86mmol, 35% over 2 steps) as a cream solid. The material was isolated as ahydrate. In addition, trace diethyl ether could not be removed undervacuum even after extended drying under vacuum.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of preventing or delaying the onset of apre-Alzheimer's cognitive dysfunction, and/or preventing or delaying theprogression of a pre-Alzheimer's condition or cognitive dysfunction toAlzheimer's disease, said method comprising: administering to a subjectcharacterized as asymptomatic, but having a known genetic risk factorfor Alzheimer's disease where said risk factor comprises an FAD mutationand/or an the APOE ε4 allele, an APP specific BACE inhibitor thatcomprises a galangin prodrug in an amount sufficient to prevent or delaythe onset of a pre-Alzheimer's cognitive dysfunction, and/or to preventor delay the progression of a pre-Alzheimer's cognitive dysfunction toAlzheimer's disease; wherein said galangin prodrug is characterized bythe formula:

wherein: R¹, R², and R³ are H, or a protecting group that is removed invivo in a mammal, wherein at least one of R¹, R², and R³ is not H; andwherein, when R¹, R², or R³ is a protecting group, said protecting groupis selected from the group consisting of

and wherein said administration produces a reduction in the CSF oflevels of one or more components selected from the group consisting oftotal-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Aβ40, pTau/Aβ42ratio and tTau/Aβ42 ratio, and/or an increase in the CSF of levels ofone or more components selected from the group consisting of Aβ42/Aβ40ratio, Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, andsAPPα/Aβ42 ratio.
 2. A method of reducing the rate of progression ofamyloidogenesis in Alzheimer's disease, said method comprising:administering to a subject in need thereof an APP specific BACEinhibitor (ASBI) that comprises a galangin prodrug in an amountsufficient to reduce the rate of progression of amyloidogenesis inAlzheimer's disease; wherein said galangin prodrug is characterized bythe formula:

wherein: R¹, R², and R³ are H, or a protecting group that is removed invivo in a mammal, wherein at least one of R¹, R², and R³ is not H; andwherein, when R¹, R², or R³ is a protecting group, said protecting groupis selected from the group consisting of

and wherein said galangin prodrug is effective to cross the blood-brainbarrier, and wherein said administration produces a reduction in the CSFof levels of one or more components selected from the group consistingof total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Aβ40, pTau/Aβ42ratio and tTau/Aβ42 ratio, and/or an increase in the CSF of levels ofone or more components selected from the group consisting of Aβ42/Aβ40ratio, Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, andsAPPα/Aβ42 ratio.
 3. The method of claim 1, wherein said APP specificBACE inhibitor is administered in a pharmaceutical formulation whereinsaid ASBI is the principle active component.
 4. The method of claim 2,wherein said APP specific BACE inhibitor is administered in apharmaceutical formulation wherein said ASBI is the principle activecomponent.
 5. The APP specific BACE inhibitor of claim 1, wherein atleast one of R¹, R², and R³ is


6. The APP specific BACE inhibitor of claim 1, wherein at least one ofR¹, R², and R³ is


7. The APP specific BACE inhibitor of claim 1, wherein at least one ofR¹, R², and R³ is


8. The APP specific BACE inhibitor of claim 1, wherein at least one ofR¹, R², and R³ is


9. The method of claim 1, wherein said known genetic risk compriseshaving the APOE ε4 allele.
 10. The method of claim 1, wherein said knowngenetic risk comprises mutations at one or more of positions 717, 670,and 671 in the APP gene.
 11. The method of claim 1, wherein said knowngenetic risk comprises relatives of said subject that have beendiagnosed with Alzheimer's disease.